Therapeutic regimen for hypertension

ABSTRACT

The invention relates to improved methods, devices, and kits for identifying and implementing an appropriate treatment regimen for subjects suffering from hypertension.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Filing under 35 U.S.C. § 371from International Application No. PCT/US2015/032651, filed May 27,2015, and published as WO 2015/183938 on Dec. 3, 2015, which applicationclaims benefit of the priority filing date of U.S. ProvisionalApplication Ser. No. 62/004,460, filed May 29, 2014, the contents ofwhich applications are specifically incorporated herein by reference intheir entireties.

BACKGROUND

Hypertension (high blood pressure) is one of the most significantpreventable contributors to disease and death in the world andrepresents the most common condition seen in the primary care setting(Kearney et al., Lancet 365:217-223 (2005)). According to the AmericanHeart Association, approximately 78 million adults (1 in 3) living inthe United States have hypertension with more than 5 million newdiagnoses made each year. Of these individuals, 82% are aware they haveit, 75% are currently being treated for it, but only 52% have theirblood pressure under control (thus, ˜48% do not have adequate bloodpressure control).

Hypertension can lead to myocardial infarction (heart attack), stroke,renal failure, and death if not detected early and treatedappropriately. In 2009, high blood pressure was listed as a primary orcontributing cause of death in about 350,000 of the approximate 2.4million U.S. deaths (14% of all deaths). From 1999-2009 the number ofdeaths attributable to hypertension increased by 44%.

Refractory (or resistant) hypertension is defined as blood pressure thatremains above clinical guideline goals in spite of concurrent use ofthree antihypertensive agents of different classes. Critically,refractory hypertension is noted in approximately 25-30% of allindividuals being treated for hypertension. Refractory hypertension is acommon clinical problem which contributes to the high levels ofmorbidity and mortality. In 2009, the direct and indirect economicburden on the United States health care system associated withhypertension was estimated at $51 billion.

Globally, nearly 1 billion individuals have been diagnosed withhypertension, with an estimate of an additional 400 million living withundiagnosed hypertension. Hypertension is the leading cause of prematuredeath and the leading cause of cardiovascular disease worldwide. Similarto the continued upward trend in prevalence as seen in the UnitedStates, it is estimated that in 2025 about 1.56 billion adults will beliving with hypertension. Because nearly two-thirds of the people livingwith hypertension worldwide reside in developing countries, providingoptimal treatment at the lowest cost is critically important.

Unfortunately, despite a significant impulse in the medical community tomove towards an “individualized medicine” approach to patient centeredtreatment, the current clinical treatment strategy is based on a setalgorithm which does not take into account individual patientdifferences. Rather, physicians are guided to choose a drug (one out ofmany options) in a given class of drugs and use that specific drug as a“first line therapy” (typically initiating with the diuretic class) andtitrate that specific drug of choice to therapeutic dosage regardless ofefficacy. It is only after a prolonged course of treatment with thatspecific class of drug that clinical efficacy is determined (typicallythree months). At this stage, if clinical guideline goals for bloodpressure have not been met, it is often recommended that the patientremain on the “first line therapy” whilst an additional drug from adifferent class of drugs (typically an Angiotensin converting enzymeinhibitor (ACE inhibitor) or Angiotensin II receptor blocker (ARB)) isadded to the pharmacologic regimen. Again, this drug is titrated torecommended therapeutic dosage and another prolonged course of treatmentis initiated before clinical efficacy is determined (an additional threemonths—six months since initiation of treatment). If at this point,clinical guideline goals for blood pressure have not been met, a thirddrug from a third class of drugs (typically a beta-blocker) is added andthe process is repeated (another three months—nine months frominitiation of treatment). Further, if clinical guideline goals havecontinued to be elusive, the diagnosis of refractory hypertension isadded and the process is reinitiated with a different combination ofdrugs, different classes of drugs, different drug options within a givenclass of drugs, different dosages, or all of the above. Thus, from thetime of initial diagnosis and the start of treatment to the point inwhich blood pressure is adequately controlled may take anywhere fromthree months to well over one year. This trial-and-error standard ofcare is clearly not optimal.

SUMMARY

The invention relates to improved methods, devices, and kits foridentifying and implementing an appropriate treatment regimen forsubjects suffering from hypertension. The methods, devices, and kitscomprehensively assess common genetic variants in the cardiac, vascular,and renal systems in an effort to improve therapeutic guidance for highblood pressure treatment. Detection of an individual's genetic variantspermits selection appropriate drug classes for that individual.Clinicians can then guide blood pressure therapy using knowledge that isspecific to their individual patient, rather than the currently employed“trial-and-error” procedures that are based on population data and useof drugs with the least initial side effects.

One aspect of the invention is a method that includes:

-   -   (a) administering a loop diuretic to a subject as a first line        therapy, without a beta blocker and without a vasodilator, if        the subject's genome comprises a WNK1 nucleic acid with a        cytosine at the variable position of rs1159744 or rs2107614;    -   (b) administering a hydrochlorothiazide to a subject as a first        line therapy, without a beta blocker and without a vasodilator,        if the subject's genome comprises an ADD1 nucleic acid with a        thymine at the variable position of rs4961, or if the test        sample comprises a SLC12A3 nucleic acid with a thymine at the        variable position of rs1529927; or    -   (c) administering a hydrochlorothiazide to a subject as a first        line therapy, without a beta blocker and without a vasodilator,        if the subject's genome comprises an ADD1 nucleic acid with a        thymine at the variable position of rs4961, or if the test        sample comprises a SLC12A3 nucleic acid with a thymine at the        variable position of rs1529927.

Another aspect of the invention is a method that includes: administeringa beta-blocker drug to a subject as a first line therapy, without adiuretic and without a hydrochlorothiazide, if the subject's genome doesnot comprise:

-   -   (a) a WNK1 nucleic acid with a cytosine at the variable position        of rs1159744;    -   (b) a WNK1 nucleic acid with a cytosine at the variable position        of rs2107614;    -   (c) an ADD1 nucleic acid with a thymine at the variable position        of rs4961; or    -   (d) a SLC12A3 nucleic acid with a thymine at the variable        position of rs1529927

but the subject's genome does comprise:

-   -   1. a CYP2D6 nucleic acid with an adenine at the variable        position of Rs3892097;    -   2. an ADRB1 nucleic acid with a cytosine at the variable        position of rs1801253;    -   3. an ADRB1 nucleic acid with an adenine at the variable        position of rs1801252;    -   4. an ADRB2 nucleic acid with a guanine at the variable position        of rs1042714; or    -   5. an ADRB2 nucleic acid with a guanine at the variable position        of rs1042713.

Another aspect of the invention is a method that includes: administeringan angiotensin II receptor blocker to a subject as a first line therapy,without a diuretic, without a hydrochlorothiazide, and without abeta-blocker, if the subject's genome does not comprise:

-   -   (a) a WNK1 nucleic acid with a cytosine at the variable position        of rs1159744;    -   (b) a WNK1 nucleic acid with a cytosine at the variable position        of rs2107614;    -   (c) an ADD1 nucleic acid with a thymine at the variable position        of rs4961; or    -   (d) a SLC12A3 nucleic acid with a thymine at the variable        position of rs1529927

but the subject's genome does comprise:

-   -   1. a renin nucleic acid with a cytosine at the variable position        of rs12750834; or    -   2. an AGT1R nucleic acid with a cytosine at the variable        position of rs5186.

Another aspect of the invention is a method that includes: administeringan ACE inhibitor to a subject without an angiotensin II receptor blockeras a first line therapy, without a diuretic, without ahydrochlorothiazide, and without a beta-blocker, if the subject's genomedoes not comprise:

-   -   (a) WNK1 nucleic acid with a cytosine at the variable position        of rs1159744;    -   (b) a WNK1 nucleic acid with a cytosine at the variable position        of rs2107614;    -   (c) an ADD1 nucleic acid with a thymine at the variable position        of rs4961; or    -   (d) a SLC12A3 nucleic acid with a thymine at the variable        position of rs1529927

but the subject's genome does comprise:

-   -   1. an ACE nucleic acid with a deletion in rs1799752; or    -   2. an AGT nucleic acid with a cytosine at the variable position        of rs699.

Another aspect of the invention is a method that includes: administeringan amiloride as a first line therapy to a subject without an ACEinhibitor, without an angiotensin II receptor blocker, without adiuretic, without a hydrochlorothiazide, and without a beta-blocker, ifthe subject's genome does not comprise:

-   -   1. a WNK1 nucleic acid with a cytosine at the variable position        of rs1159744;    -   2. a WNK1 nucleic acid with a cytosine at the variable position        of rs2107614;    -   3. an ADD1 nucleic acid with a thymine at the variable position        of rs4961; or    -   4. a SLC12A3 nucleic acid with a thymine at the variable        position of rs1529927.    -   but if the subject's genome does comprise a SCNN1A nucleic acid        with an adenine at the variable position of rs2228576.

The methods can also include administering a second line therapy drugafter administration of the first line therapy for at least 1 month,wherein the second line therapy drug is selected from the groupconsisting of diuretic, a beta-blocker, an ACE inhibitor, a vasodilator,and a combination thereof.

Devices, compositions, methods, and kits are also described herein foridentifying and implementing an appropriate treatment regimen forsubjects suffering from hypertension.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram illustrating the interplay between theheart, blood vessels, and kidney in blood pressure regulation.

FIG. 2 is a schematic diagram illustrating of the types of genes usefulfor evaluating hypertension, and representative single nucleotidepolymorphisms that are correlated with blood pressure drug responses.

FIG. 3A-3B are schematic diagrams illustrating processing of testsamples. For example, each subject can collect two swabs. The A swab cancollect cell material from the inside of the right cheek, while the Bswab can collect cell material from the left cheek. For FIG. 3A, the Aswab can be the initial swab entered into the process (from DNAExtraction to Reporting). If the A swab fails, during DNA Yield andPurity Analysis, Genetic analysis, or the PCR QA Assay then the B swabcan be entered into the system as illustrated. FIG. 3B shows the sameprocess but without the steps of DNA Stocks Storage and Future Testingif the sample Passes Yield and Purity Assays.

FIG. 4 is a schematic diagram illustrating handling of DNA samples.

FIG. 5 is a schematic diagram illustrating processing of the sampleafter PCR amplification.

FIG. 6 illustrates alignment of sample results to a human referencesequence using the CLC DNA workbench program for creating an alignmentfile from which the allele call is made and added to the final SNP callreport (SEQ ID NOs:82-85).

FIG. 7 is an example of 2% agarose gel used to score the presence orabsence of a 288 bp ALU by visually examining the gel for either thehigher molecular weight band (indicating the presence of the 288 bpALU), the lower molecular weight band (indicating the absence of the 288bp ALU), or both (indicating a heterozygous state).

FIG. 8 is a bar graph of urinary sodium output as a function of geneticvariation of SCNN1A.

FIG. 9 is a bar graph of mean arterial blood pressure as a function ofgenetic variation of SCNN1A.

DETAILED DESCRIPTION

Methods, devices, and kits are described herein for selectingindividualized hypertension treatment regimens that are more effectivethan currently available regimens. The methods, devices, and kitsinclude assays for identifying genetic variants in individual subjectsthat make the individual more or less responsive to specificmedications. When the appropriate medication is administered, thesubject's blood pressure is appropriately controlled, and side effectsare avoided. Genetic variants present in genes such as ADRB1, ADRB2,CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen,angiotensin receptor, renin, Na⁺ channels (such as SCNN1A), adducin,sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/orWNK1 genes are correlated with heightened or reduced responsiveness tovarious blood pressure medications. Although there are a number ofhypertension drugs available on the market, subjects react differentlyto such drugs. The kits, methods, and devices described herein areuseful for detecting which subjects benefit from which therapeuticregimen.

High Blood Pressure (Hypertension)

The development of high blood pressure in humans is the result of one ormore of three physiologic maladaptations: 1) elevated cardiac output(liters of blood ejected from the heart per minute) that increases theamount of blood pressing against the vessels, 2) relatively narrow bloodvessels that results in increased pressure towards the lumen of theblood vessel due to Poiseuille's Law (which describes the inverserelationship between the diameter of a tube (vessel) and the pressureagainst the walls of the tube (vessel), all else being equal), or 3)increased sodium (Na⁺) absorption in the kidney which results inincreased blood volume (and the amount of blood pumped per minute,cardiac output) and subsequently increased outward pressure against thetubes (vessels).

Blood pressure is highly regulated and tightly controlled in humans,such that in the event of a drop in cardiac output, the heart sends asignal to the kidneys (via the proteins atrial natriuretic peptide andbrain-type natriuretic peptide, among others) and vessels with theultimate function of increasing Na⁺ reabsorption to increase plasmavolume and vasoconstriction to increase blood pressure. Similarly, ifthere is a drop in blood pressure, there is an increase in cardiacoutput (primarily via an immediate increase in heart rate throughinhibition of the parasympathetic nervous system) to compensate and anincrease in the renin-angiotensin aldosterone system which results inboth constriction of blood vessels (which increases blood pressure) andan increase in Na⁺ and, therefore, fluid retention in the kidney, whichincreases plasma volume and can increase blood pressure. Hence, thehuman body provides redundant functions to maintain blood pressure bothin the short-term and in the long-term, by regulating the interplaybetween the heart, blood vessels, and kidney. FIG. 1 is a schematicdiagram illustrating the interplay between the heart, blood vessels, andkidneys involved in regulating blood pressure.

Blood pressure therapy following diagnosis traditionally follows aregimented algorithm based on therapies effective across the generalpopulation. Currently, clinicians start a patient who has been diagnosedwith high blood pressure on a diuretic (to reduce renal Na⁺reabsorption). If such a diuretic does not work within a period of time,the clinician next tries a vasodilator, and if this is not effective,then a clinician will lastly prescribe a beta-blocker. Thistrial-and-error process to lower a patient's blood pressure can takeseveral months, is costly, and threatens the health of the patientbecause the patient's hypertension is frequently not adequatelycontrolled in a timely manner.

Such currently available methods are in stark contrast to the methods,devices, and kits described herein that involve specifically testing anindividual's genetic profile and, as illustrated herein, basingtherapeutic treatment on the results of such testing. Hence, themethods, devices, and kits described herein for evaluating a bloodpressure genetic panel to improve treatment of hypertensive subjects, byquickly identifying more effective medications, thereby avoiding sideeffects and delays in treatment. Applicants' methods are therefore animprovement over the currently available trial-and-error procedures thatfrequently result in side effects and delays in effective treatment.

Ranking of Genotypes that Affect Therapy

The genotype of a subject can significantly affect the response of thesubject to blood pressure medications because certain functionalpolymorphisms have greater effects on the physiology of a subject thanothers. The following is a summary of polymorphisms in order of theirimpact on blood pressure and which drugs should be employed by subjectswith such genetic variations.

-   -   1. Subjects with the WNK1 polymorphism defined by rs1159744 (SEQ        ID NO:34, with cytosine at the variable position), benefit more        from loop diuretics.    -   2. Subjects with the WNK1 polymorphism defined by rs2107614 (SEQ        ID NO:33, with cytosine at the variable position), benefit more        from loop diuretics.    -   3. Subjects with the ADD1 polymorphism defined by rs4961 (SEQ ID        NO:27, with thymine at the variable position), benefit more from        hydrochlorothiazides.    -   4. Subjects with the SLC12A3 polymorphism defined by rs1529927        (SEQ ID NO:30, with thymine at the variable position), benefit        more from hydrochlorothiazides.        Any homozygous combination at one or more of the WNK1 Rs1159744,        WNK1 Rs2107614, ADD1 Rs4961, and SLC12A3 rs1529927 polymorphisms        means that diuretics should be the first-line therapy,        especially if the patient is heterozygous for polymorphisms in        genes responsive to beta-blockers or vasodilators.    -   5. Subjects with the CYP2D6 polymorphism defined by Rs3892097        (SEQ ID NO:10, with adenine at the variable position), plus the        ADRB1 polymorphism defined by rs1801253 (SEQ ID NO:3, with        cytosine as the variable nucleotide), plus the ADRB1        polymorphism defined by rs1801252 (SEQ ID NO:2, with adenine as        the variable nucleotide) benefit from beta-blockade classes of        drugs.    -   6. Subjects with the renin polymorphism defined by rs12750834        (SEQ ID NO:20, with cytosine as the variable nucleotide) plus        the AGT1R polymorphism defined by rs5186 (SEQ ID NO:16, with        cytosine as the variable nucleotide), which affect responses to        angiotensin II receptor blockers.    -   7. Subjects with the ACE deletion defined by Rs1799752 (SEQ ID        NO:35) and the AGT polymorphism defined by rs699 (SEQ ID NO:14,        with cytosine as the variable nucleotide) can benefit from the        combination of an angiotensin II receptor blocker and an ACE        inhibitor.    -   8. Subjects with the SCNN1A polymorphism defined by rs2228576        (SEQ ID NO:22, with adenine as the variable position) can        benefit from administration of amiloride.    -   9. Subjects with the ADRB2 polymorphism defined by rs1042714        (SEQ ID NO:7, with guanine as the variable nucleotide) can        benefit from administration of a non-selective beta-blockade.    -   10. Subjects with the ADRB2 polymorphism defined by s1042713        (SEQ ID NO:6, with guanine as the variable nucleotide) can        benefit from administration of a non-selective beta-blockade.        All patients do not respond to same. Some subjects have        genotypes that can significantly affect their response to        medications. When clinicians employ currently available        procedures (diuretic first, then vasodilator, then beta        blocker), some patients will benefit but others will not respond        or will respond negatively. Hence, some patients would benefit        from initial administration of a vasodilator or a beta-blocker,        rather than a diuretic.        Beta-Blocker Responsive Polypeptides and Nucleic Acids

There are two primary receptors within the heart that influence bothheart rate (chronotropic effect) and heart contractility (inotrpiceffect) (Brodde, Am J Cardiol 62:24C-29C (1988), the beta-1 adrenergicreceptors (β₁AR, encoded by the ADRB1 gene) and the beta-2 adrenergicreceptors (β₂AR, encoded by the ADRB2 gene).

The heart is primary comprised of beta-1 adrenergic receptors, which arelocated on 80% of the ventricular wall surface, 70% of the atrial wallsurface, and 95% of the sino-atrial (SA) node. The atria of the heartreceive blood that returns from the body (right atria) of lungs (leftatria) whereas the ventricles pump blood to the lungs (right ventricle)and body (left ventricle). The sino-atrial node primarily controls heartrate.

Although heart rate and cardiac contractility are primarily regulated byβ₁AR, the β₂AR also play a role, primarily in cardiac contractility.Stimulation of either β₁AR or β₂AR can influence heart rate and cardiaccontractility through increases in intracellular c-AMP and proteinkinase A (PKA) which, ultimately, alter Ca⁺-channel sensitivity andreduce the threshold needed for an action potential. Therefore, cardiacoutput (and blood pressure) can be increased through increases in β₁ARand/or β₂AR activities. If a variant β₁AR or β₂AR gene encodes a morefunctional receptor, cardiac output is increased.

β₁AR and β₂AR activities can be modulated through the use of selective(e.g., atenolol and metoprolol) and non-selective (e.g., propranolol andcarvedilol) beta-blockers. The selective beta-blockers are selective forinhibiting the β₁AR. The non-selective beta-blockers inhibit both β₁ARand β₂AR. Both types of beta-blockers tend to decrease blood pressurethrough a decrease in heart rate and cardiac contractility, whichultimately results in a decrease in cardiac output. Similarly, theadministration of a β₂AR-agonist (e.g., albuterol sulfate) tends toincrease cardiac output and heart rate (Snyder et al., Pharmacotherapy31:748-756 (2011)). Thus, both β₁AR and β₂AR are important in theregulation of cardiac output.

Just as stimulation of these receptors can elevate cardiac output andincrease blood pressure, so too can genetic variation of the genes thatencode β₁AR and β₂AR (ADRB1 and ADRB2) elevate receptor activity andincrease blood pressure. Conversely, some ADRB1 and ADRB2 geneticvariants encode receptors with reduced activity. In addition, some ADRB1and ADRB2 genetic variants exhibit reduced, or enhanced, responsivenessto blood pressure medications such as beta-blockers. Not all individualsrespond similarly to beta-blockade, despite similar clinical andenvironmental conditions. As described herein, the effectiveness ofbeta-blockers is dependent to some extent upon the genetic make-up ofthe subjects to which the beta-blockers are administered.

Sequences for various adrenergic receptors are available, for example,from the National Center for Biotechnology Information (see website atncbi.nlm.nih.gov).

For example, a full length human ADRB1 cDNA nucleotide sequence isavailable from the database maintained by the National Center forBiotechnology Information (see website at ncbi.nlm.nih.gov), which hasaccession number NM_00064 (GI:110349783), and is shown below as SEQ IDNO:1.

1 GCACCACGCC GCCCGGGCTT CTGGGGTGTT CCCCAACCAC 41GGCCCAGCCC TGCCACACCC CCCGCCCCCG GCCTCCGCAG 81CTCGGCATGG GCGCGGGGGT GCTCGTCCTG GGCGCCTCCG 121AGCCCGGTAA CCTGTCGTCG GCCGCACCGC TCCCCGACGG 161CGCGGCCACC GCGGCGCGGC TGCTGGTGCC CGCGTCGCCG 201CCCGCCTCGT TGCTGCCTCC CGCCAGCGAA  A GCCCCGAGC 241CGCTGTCTCA GCAGTGGACA GCGGGCATGG GTCTGCTGAT 281GGCGCTCATC GTGCTGCTCA TCGTGGCGGG CAATGTGCTG 321GTGATCGTGG CCATCGCCAA GACGCCGCGG CTGCAGACGC 361TCACCAACCT CTTCATCATG TCCCTGGCCA GCGCCGACCT 401GGTCATGGGG CTGCTGGTGG TGCCGTTCGG GGCCACCATC 441GTGGTGTGGG GCCGCTGGGA GTACGGCTCC TTCTTCTGCG 481AGCTGTGGAC CTCAGTGGAC GTGCTGTGCG TGACGGCCAG 521CATCGAGACC CTGTGTGTCA TTGCCCTGGA CCGCTACCTC 561GCCATCACCT CGCCCTTCCG CTACCAGAGC CTGCTGACGC 601GCGCGCGGGC GCGGGGCCTC GTGTGCACCG TGTGGGCCAT 641CTCGGCCCTG GTGTCCTTCC TGCCCATCCT CATGCACTGG 681TGGCGGGCGG AGAGCGACGA GGCGCGCCGC TGCTACAACG 721ACCCCAAGTG CTGCGACTTC GTCACCAACC GGGCCTACGC 761CATCGCCTCG TCCGTAGTCT CCTTCTACGT GCCCCTGTGC 801ATCATGGCCT TCGTGTACCT GCGGGTGTTC CGCGAGGCCC 841AGAAGCAGGT GAAGAAGATC GACAGCTGCG AGCGCCGTTT 881CCTCGGCGGC CCAGCGCGGC CGCCCTCGCC CTCGCCCTCG 921CCCGTCCCCG CGCCCGCGCC GCCGCCCGGA CCCCCGCGCC 961CCGCCGCCGC CGCCGCCACC GCCCCGCTGG CCAACGGGCG 1001TGCGGGTAAG CGGCGGCCCT CGCGCCTCGT GGCCCTGCGC 1041GAGCAGAAGG CGCTCAAGAC GCTGGGCATC ATCATGGGCG 1081TCTTCACGCT CTGCTGGCTG CCCTTCTTCC TGGCCAACGT 1121GGTGAAGGCC TTCCACCGCG AGCTGGTGCC CGACCGCCTC 1161TTCGTCTTCT TCAACTGGCT GGGCTACGCC AACTCGGCCT 1201TCAACCCCAT CATCTACTGC CGCAGCCCCG ACTTCCGCAA 1241 GGCCTTCCAG  GGACTGCTCT GCTGCGCGCG CAGGGCTGCC 1281CGCCGGCGCC ACGCGACCCA CGGAGACCGG CCGCGCGCCT 1321CGGGCTGTCT GGCCCGGCCC GGACCCCCGC CATCGCCCGG 1361GGCCGCCTCG GACGACGACG ACGACGATGT CGTCGGGGCC 1401ACGCCGCCCG CGCGCCTGCT GGAGCCCTGG GCCGGCTGCA 1441ACGGCGGGGC GGCGGCGGAC AGCGACTCGA GCCTGGACGA 1481GCCGTGCCGC CCCGGCTTCG CCTCGGAATC CAAGGTGTAG 1521GGCCCGGCGC GGGGCGCGGA CTCCGGGCAC GGCTTCCCAG 1561GGGAACGAGG AGATCTGTGT TTACTTAAGA CCGATAGCAG 1601GTGAACTCGA AGCCCACAAT CCTCGTCTGA ATCATCCGAG 1641GCAAAGAGAA AAGCCACGGA CCGTTGCACA AAAAGGAAAG 1681TTTGGGAAGG GATGGGAGAG TGGCTTGCTG ATGTTCCTTG 1721TTGTTTTTTT TTTCTTTTCT TTTCTTTCTT CTTCTTTTTT 1741TTTTTTTTTT TTTTTTCTGT TTGTGGTCCG GCCTTCTTTT 1801GTGTGTGCGT GTGATGCATC TTTAGATTTT TTTCCCCCAC 1841CAGGTGGTTT TTGACACTCT CTGAGAGGAC CGGAGTGGAA 1881GATGGGTGGG TTAGGGGAAG GGAGAAGCAT TAGGAGGGGA 1921TTAAAATCGA TCATCGTGGC TCCCATCCCT TTCCCGGGAA 1961CAGGAACACA CTACCAGCCA GAGAGAGGAG AATGACAGTT 2001TGTCAAGACA TATTTCCTTT TGCTTTCCAG AGAAATTTCA 2041TTTTAATTTC TAAGTAATGA TTTCTGCTGT TATGAAAGCA 2081AAGAGAAAGG ATGGAGGCAA AATAAAAAAA AATCACGTTT 2121CAAGAAATGT TAAGCTCTTC TTGGAACAAG CCCCACCTTG 2161CTTTCCTTGT GTAGGGCAAA CCCGCTGTCC CCCGCGCGCC 2201TGGGTGGTCA GGCTGAGGGA TTTCTACCTC ACACTGTGCA 2241TTTGCACAGC AGATAGAAAG ACTTGTTTAT ATTAAACAGC 2281TTATTTATGT ATCAATATTA GTTGGAAGGA CCAGGCGCAG 2321AGCCTCTCTC TGTGACATGT GACTCTGTCA ATTGAAGACA 2361GGACATTAAA AGAGAGCGAG AGAGAGAAAC AGTTCAGATT 2401ACTGCACATG TGGATAAAAA CAAAAACAAA AAAAAGGAGT 2441GGTTCAAAAT GCCATTTTTG CACAGTGTTA GGAATTACAA 2481AATCCACAGA AGATGTTACT TGCACAAAAA GAAATTAAAT 2521ATTTTTTAAA GGGAGAGGGG CTGGGCAGAT CTTAAATAAA 2561ATTCAAACTC TACTTCTGTT GTCTAGTATG TTATTGAGCT 2601AATGATTCAT TGGGAAAATA CCTTTTTATA CTCCTTTATC 2641ATGGTACTGT AACTGTATCC ATATTATAAA TATAATTATC 2681TTAAGGATTT TTTATTTTTT TTTATGTCCA AGTGCCCACG 2721TGAATTTGCT GGTGAAAGTT AGCACTTGTG TGTAAATTCT 2761ACTTCCTCTT GTGTGTTTTA CCAAGTATTT ATACTCTGGT 2801GCAACTAACT ACTGTGTGAG GAATTGGTCC ATGTGCAATA 2841AATACCAATG AAGCACAATC AA

The rs1801252 single nucleotide polymorphism (SNP) is present in theADRB1 gene, where the variable nucleotide at about position 231(underlined) can be adenine in some individuals and guanine in others.The rs1801252 sequence (SEQ ID NO:2) is shown below, where theunderlined A/G is the SNP.

CTCGTTGCTGCCTCCCGCCAGCGAA [A/G] GCCCCGAGCCGCTGTCTCAG CAGTG.

The rs1801253 single nucleotide polymorphism (SNP) is also present inthe ADRB1 gene, where the variable nucleotide at about position 1251(underlined) can be guanine in some individuals and cytosine in others.The rs1801253 sequence (SEQ ID NO:3) is shown below, where theunderlined C/G is the SNP.

CCCCGACTTCCGCAAGGCCTTCCAG [C/G] GACTGCTCTGCTGCGCGCGC AGGGC.

The β₁AR polypeptide encoded by the ADRB1 cDNA with SEQ ID NO:1 has thefollowing sequence (SEQ ID NO:4).

1 MGAGVLVLGA SEPGNLSSAA PLPDGAATAA RLLVPASPPA 41 SLLPPASE SP EPLSQQWTAG MGLLMALIVL LIVAGNVLVI 81VAIAKTPRLQ TLTNLFIMSL ASADLVMGLL VVPFGATIVV 121WGRWEYGSFF CELWTSVDVL CVTASIETLC VIALDRYLAI 161TSPFRYQSLL TRARARGLVC TVWAISALVS FLPILMHWWR 201AESDEARRCY NDPKCCDFVT NRAYAIASSV VSFYVPLCIM 241AFVYLRVFRE AQKQVKKIDS CERRFLGGPA RPPSPSPSPV 281PAPAPPPGPP RPAAAAATAP LANGRAGKRR PSRLVALREQ 321KALKTLGIIM GVFTLCWLPF FLANVVKAFH RELVPDRLFV 361FFNWLGYANS AFNPIIYCRS PDFRKAFQ G L LCCARRAARR 401RHATHGDRPR ASGCLARPGP PPSPGAASDD DDDDVVGATP 441PARLLEPWAG CNGGAAADSD SSLDEPCRPG FASESKV

Note that the underlined amino acid at position 49 is serine becausesome individuals have SEQ ID NO:1 or 2, where the variable nucleotide atabout position 231 of SEQ ID NO:1 is adenine. However, position 49 ofSEQ ID NO:4 can be glycine in some individuals because those individualhave guanine at nucleotide position 231 in SEQ ID NO:1.

Note also that the glycine at position 389 is an arginine (instead ofglycine) as shown for SEQ ID NO:4 when position 1251 of SEQ ID NO:1 is acytosine.

Individuals with serine at β₁AR amino acid position 49 and/or arginineat position 389 are more responsive to beta-blockers than those withglycines at these positions. Hence, for example, an individual whoexpresses the β₁AR polypeptide with SEQ ID NO:4, will be more responsiveto beta-blockers than an individual who expresses the β₁AR polypeptidewith glycines at both positions 49 and 389.

A full length human ADRB2 cDNA nucleotide sequence is available from thedatabase maintained by the National Center for Biotechnology Information(see website at ncbi.nlm.nih.gov), which has accession number NM_000024(GI:283483994), and is shown below as SEQ ID NO:5.

1 GCACATAACG GGCAGAACGC ACTGCGAAGC GGCTTCTTCA 41GAGCACGGGC TGGAACTGGC AGGCACCGCG AGCCCCTAGC 81ACCCGACAAG CTGAGTGTGC AGGACGAGTC CCCACCACAC 121CCACACCACA GCCGCTGAAT GAGGCTTCCA GGCGTCCGCT 161CGCGGCCCGC AGAGCCCCGC CGTGGGTCCG CCCGCTGAGG 201CGCCCCCAGC CAGTGCGCTC ACCTGCCAGA CTGCGCGCCA 241TGGGGCAACC CGGGAACGGC AGCGCCTTCT TGCTGGCACC 281 CAAT AGAAGC CATGCGCCGG ACCACGACGT CACGCAG C AA 321AGGGACGAGG TGTGGGTGGT GGGCATGGGC ATCGTCATGT 361CTCTCATCGT CCTGGCCATC GTGTTTGGCA ATGTGCTGGT 401CATCACAGCC ATTGCCAAGT TCGAGCGTCT GCAGACGGTC 441ACCAACTACT TCATCACTTC ACTGGCCTGT GCTGATCTGG 481TCATGGGCCT GGCAGTGGTG CCCTTTGGGG CCGCCCATAT 521TCTTATGAAA ATGTGGACTT TTGGCAACTT CTGGTGCGAG 561TTTTGGACTT CCATTGATGT GCTGTGCGTC ACGGCCAGCA 601TTGAGACCCT GTGCGTGATC GCAGTGGATC GCTACTTTGC 641CATTACTTCA CCTTTCAAGT ACCAGAGCCT GCTGACCAAG 681AATAAGGCCC GGGTGATCAT TCTGATGGTG TGGATTGTGT 721CAGGCCTTAC CTCCTTCTTG CCCATTCAGA TGCACTGGTA 761CCGGGCCACC CACCAGGAAG CCATCAACTG CTATGCCAAT 801GAGACCTGCT GTGACTTCTT CACGAACCAA GCCTATGCCA 841TTGCCTCTTC CATCGTGTCC TTCTACGTTC CCCTGGTGAT 881CATGGTCTTC GTCTACTCCA GGGTCTTTCA GGAGGCCAAA 921AGGCAGCTCC AGAAGATTGA CAAATCTGAG GGCCGCTTCC 961ATGTCCAGAA CCTTAGCCAG GTGGAGCAGG ATGGGCGGAC 1001GGGGCATGGA CTCCGCAGAT CTTCCAAGTT CTGCTTGAAG 1041GAGCACAAAG CCCTCAAGAC GTTAGGCATC ATCATGGGCA 1081CTTTCACCCT CTGCTGGCTG CCCTTCTTCA TCGTTAACAT 1121TGTGCATGTG ATCCAGGATA ACCTCATCCG TAAGGAAGTT 1161TACATCCTCC TAAATTGGAT AGGCTATGTC AATTCTGGTT 1201TCAATCCCCT TATCTACTGC CGGAGCCCAG ATTTCAGGAT 1241TGCCTTCCAG GAGCTTCTGT GCCTGCGCAG GTCTTCTTTG 1281AAGGCCTATG GGAATGGCTA CTCCAGCAAC GGCAACACAG 1321GGGAGCAGAG TGGATATCAC GTGGAACAGG AGAAAGAAAA 1361TAAACTGCTG TGTGAAGACC TCCCAGGCAC GGAAGACTTT 1401GTGGGCCATC AAGGTACTGT GCCTAGCGAT AACATTGATT 1441CACAAGGGAG GAATTGTAGT ACAAATGACT CACTGCTGTA 1481AAGCAGTTTT TCTACTTTTA AAGACCCCCC CCCCCAACAG 1521AACACTAAAC AGACTATTTA ACTTGAGGGT AATAAACTTA 1561GAATAAAATT GTAAAATTGT ATAGAGATAT GCAGAAGGAA 1601GGGCATCCTT CTGCCTTTTT TATTTTTTTA AGCTGTAAAA 1641AGAGAGAAAA CTTATTTGAG TGATTATTTG TTATTTGTAC 1681AGTTCAGTTC CTCTTTGCAT GGAATTTGTA AGTTTATGTC 1721TAAAGAGCTT TAGTCCTAGA GGACCTGAGT CTGCTATATT 1761TTCATGACTT TTCCATGTAT CTACCTCACT ATTCAAGTAT 1801TAGGGGTAAT ATATTGCTGC TGGTAATTTG TATCTGAAGG 1841AGATTTTCCT TCCTACACCC TTGGACTTGA GGATTTTGAG 1881TATCTCGGAC CTTTCAGCTG TGAACATGGA CTCTTCCCCC 1921ACTCCTCTTA TTTGCTCACA CGGGGTATTT TAGGCAGGGA 1961TTTGAGGAGC AGCTTCAGTT GTTTTCCCGA GCAAAGTCTA 2001AAGTTTACAG TAAATAAATT GTTTGACCAT GCCTTCATTG 2041 CAAAAAAAAA AAAAAAAA

The rs1042713 single nucleotide polymorphism (SNP) is present in theADRB2 gene, where the variable nucleotide at about position 285(underlined) can be in adenine some individuals and guanine in others.The rs1042713 sequence (SEQ ID NO:6) is shown below, where theunderlined A/G is the SNP.

CAGCGCCTTCTTGCTGGCACCCAAT [A/G] GAAGCCATGCGCCGGACCAC GACGT.

The rs1042714 single nucleotide polymorphism (SNP) is also present inthe ADRB2 gene, where the variable nucleotide at about position 318(underlined) can be cytosine in some individuals and guanine in others.The rs1042714 sequence (SEQ ID NO:7) is shown below, where theunderlined C/G is the SNP.

TGCGCCGGACCACGACGTCACGCAG [C/G] AAAGGGACGAGGTGTGGGTG GTGGG.

The β₂AR polypeptide encoded by the ADRB2 cDNA with SEQ ID NO:5 has thefollowing sequence (SEQ ID NO:8).

1 MGQPGNGSAF LLAPN R SHAP DHDVTQ Q RDE VWVVGMGIVM 41SLIVLAIVFG NVLVITAIAK FERLQTVTNY FITSLACADL 81VMGLAVVPFG AAHILMKMWT FGNFWCEFWT SIDVLCVTAS 121IETLCVIAVD RYFAITSPFK YQSLLTKNKA RVIILMVWIV 161SGLTSFLPIQ MHWYRATHQE AINCYANETC CDFFTNQAYA 201IASSIVSFYV PLVIMVFVYS RVFQEAKRQL QKIDKSEGRF 241HVQNLSQVEQ DGRTGHGLRR SSKFCLKEHK ALKTLGIIMG 281TFTLCWLPFF IVNIVHVIQD NLIRKEVYIL LNWIGYVNSG 321FNPLIYCRSP DFRIAFQELL CLRRSSLKAY GNGYSSNGNT 361GEQSGYHVEQ EKENKLLCED LPGTEDFVGH QGTVPSDNID 401 SQGRNCSTND SLL

Note that the underlined arginine at position 16 of SEQ ID NO:8 isarginine because some individuals have nucleotide sequence SEQ ID NO:5,where the nucleotide at about position 285 is adenine. However, position16 of SEQ ID NO:8 can be glycine in some individuals because thoseindividuals have guanine at nucleotide position 285 in SEQ ID NO:5.

Note also that the glutamine at position 27 of SEQ ID NO:8 is a glutamicacid when position 318 of nucleotide sequence SEQ ID NO:5 is a guanine.

Individuals with glycine at position 16 and/or glutamic acid at β₂ARposition 27 are more responsive to beta-blockers than those witharginine and glutamine, respectively, at these positions. Hence, forexample, an individual who expresses the β₂AR polypeptide with SEQ IDNO:5, will be more responsive to beta-blockers than an individual whoexpresses the β₂AR polypeptide with arginine and glutamine at positions16 and 27.

The gene that encodes cytochrome P450 2D6 (CYP2D6) has been shown toalter the metabolism of the drugs in the beta-blocker class. Thisalteration in drug metabolism can alter the amount of bioavailable drug.Poor drug metabolizers tend to have more drugs available in the body forlonger and will, therefore, have a greater response to therapy. Incontrast, active metabolizers of a drug will have less of the drugavailable in their system and will respond poorly to therapy.

Because of the importance of CYP2D6 on beta-blocker metabolism, thisgene is a useful marker of responsive to beta-blocker therapy.

A full length human CYP2D6 cDNA nucleotide sequence is available fromthe database maintained by the National Center for BiotechnologyInformation (see website at ncbi.nlm.nih.gov), which has accessionnumber NM_000106.5 (GI:392513720), and is shown below as SEQ ID NO:9.

   1 GTGCTGAGAG TGTCCTGCCT GGTCCTCTGT GCCTGGTGGG   41GTGGGGGTGC CAGGTGTGTC CAGAGGAGCC CATTTGGTAG   81TGAGGCAGGT ATGGGGCTAG AAGCACTGGT GCCCCTGGCC  121GTGATAGTGG CCATCTTCCT GCTCCTGGTG GACCTGATGC  161ACCGGCGCCA ACGCTGGGCT GCACGCTACC CACCAGGCCC  201CCTGCCACTG CCCGGGCTGG GCAACCTGCT GCATGTGGAC  241TTCCAGAACA CACCATACTG CTTCGACCAG TTGCGGCGCC  281GCTTCGGGGA CGTGTTCAGC CTGCAGCTGG CCTGGACGCC  321GGTGGTCGTG CTCAATGGGC TGGCGGCCGT GCGCGAGGCG  361CTGGTGACCC ACGGCGAGGA CACCGCCGAC CGCCCGCCTG  401TGCCCATCAC CCAGATCCTG GGTTTCGGGC CGCGTTCCCA  441AGGGGTGTTC CTGGCGCGCT ATGGGCCCGC GTGGCGCGAG  481CAGAGGCGCT TCTCCGTGTC CACCTTGCGC AACTTGGGCC  521TGGGCAAGAA GTCGCTGGAG CAGTGGGTGA CCGAGGAGGC  561CGCCTGCCTT TGTGCCGCCT TCGCCAACCA CTCC G GACGC  601CCCTTTCGCC CCAACGGTCT CTTGGACAAA GCCGTGAGCA  641ACGTGATCGC CTCCCTCACC TGCGGGCGCC GCTTCGAGTA  681CGACGACCCT CGCTTCCTCA GGCTGCTGGA CCTAGCTCAG  721GAGGGACTGA AGGAGGAGTC GGGCTTTCTG CGCGAGGTGC  761TGAATGCTGT CCCCGTCCTC CTGCATATCC CAGCGCTGGC  801TGGCAAGGTC CTACGCTTCC AAAAGGCTTT CCTGACCCAG  841CTGGATGAGC TGCTAACTGA GCACAGGATG ACCTGGGACC  881CAGCCCAGCC CCCCCGAGAC CTGACTGAGG CCTTCCTGGC  921AGAGATGGAG AAGGCCAAGG GGAACCCTGA GAGCAGCTTC  961AATGATGAGA ACCTGCGCAT AGTGGTGGCT GACCTGTTCT 1001CTGCCGGGAT GGTGACCACC TCGACCACGC TGGCCTGGGG 1041CCTCCTGCTC ATGATCCTAC ATCCGGATGT GCAGCGCCGT 1081GTCCAACAGG AGATCGACGA CGTGATAGGG CAGGTGCGGC 1121GACCAGAGAT GGGTGACCAG GCTCACATGC CCTACACCAC 1161TGCCGTGATT CATGAGGTGC AGCGCTTTGG GGACATCGTC 1201CCCCTGGGTG TGACCCATAT GACATCCCGT GACATCGAAG 1241TACAGGGCTT CCGCATCCCT AAGGGAACGA CACTCATCAC 1281CAACCTGTCA TCGGTGCTGA AGGATGAGGC CGTCTGGGAG 1321AAGCCCTTCC GCTTCCACCC CGAACACTTC CTGGATGCCC 1361AGGGCCACTT TGTGAAGCCG GAGGCCTTCC TGCCTTTCTC 1401AGCAGGCCGC CGTGCATGCC TCGGGGAGCC CCTGGCCCGC 1441ATGGAGCTCT TCCTCTTCTT CACCTCCCTG CTGCAGCACT 1481TCAGCTTCTC GGTGCCCACT GGACAGCCCC GGCCCAGCCA 1521CCATGGTGTC TTTGCTTTCC TGGTGAGCCC ATCCCCCTAT 1561GAGCTTTGTG CTGTGCCCCG CTAGAATGGG GTACCTAGTC 1601CCCAGCCTGC TCCCTAGCCA GAGGCTCTAA TGTACAATAA 1641AGCAATGTGG TAGTTCCAAA AAAAAAAAAA AAA

The rs3892097 single nucleotide polymorphism (SNP) is present in theCYP2D6 gene, where the variable nucleotide at a splice site at aboutposition 595 in SEQ ID NO:9 (underlined), which can be in adenine someindividuals and guanine in others.

The rs3892097 sequence (SEQ ID NO:10) of the CYP2D6 gene is shown below,where the underlined A/G is the SNP.

CCCTTACCCGCATCTCCCACCCCCA[A/G]GACGCCCCTTTCGCCCCAAC GGTCT.Because the SNP occurs near a splice site, the sequences to the left ofthe SNP site in SEQ ID NO:10 do not appear in the SEQ ID NO:9 nucleotideCYP2D6 cDNA sequence.

The cytochrome P450 2D6 polypeptide encoded by the CYP2D6 cDNA with SEQID NO:9 has the following sequence (SEQ ID NO:11).

  1 MGLEALVPLA VIVAIFLLLV DLMHRRQRWA ARYPPGPLPL  41PGLGNLLHVD FQNTPYCFDQ LRRRFGDVFS LQLAWTPVVV  81LNGLAAVREA LVTHGEDTAD RPPVPITQIL GFGPRSQGVF 121LARYGPAWRE QRRFSVSTLR NLGLGKKSLE QWVTEEAACL 161 CAAFANHS GR PFRPNGLLDK AVSNVIASLT CGRRFEYDDP 201RFLRLLDLAQ EGLKEESGFL REVLNAVPVL LHIPALAGKV 241LRFQKAFLTQ LDELLTEHRM TWDPAQPPRD LTEAFLAEME 281KAKGNPESSF NDENLRIVVA DLFSAGMVTT STTLAWGLLL 321MILHPDVQRR VQQEIDDVIG QVRRPEMGDQ AHMPYTTAVI 361HEVQRFGDIV PLGVTHMTSR DIEVQGFRIP KGTTLITNLS 401SVLKDEAVWE KPFRFHPEHF LDAQGHFVKP EAFLPFSAGR 441RACLGEPLAR MELFLFFTSL LQHFSFSVPT GQPRPSHHGV 481 FAFLVSPSPY ELCAVPR

Note that the underlined glycine at position 169 of SEQ ID NO:11 isglycine because some individuals have nucleotide sequence SEQ ID NO:9,where the nucleotide at about position 595 is guanine. However, position169 of SEQ ID NO:11 can be arginine in some individuals because thoseindividuals have adenine at nucleotide position 295 in SEQ ID NO:9.

A patient with that is homozygous for adenine (AA) at the rs3892097variable site will express CYP2D6 with arginine at position 169 and willnot metabolize metoprolol and propranolol as quickly as those withguanine (glycine). Hence, homozygous individuals with adenine (AA) atthe rs3892097 variable site have higher plasma levels of metoprolol andpropranolol after taking these drugs than subjects that are nothomozygous for adenine (AA) at the rs3892097 variable site. Homozygousindividuals with adenine (AA) at the rs3892097 variable site wouldrespond more normally to atenolol and carvedilol, which do not requireCYP2D6 for their metabolism.

Vasodilation Therapy

Dilation of blood vessels results in decreases in blood pressure,whereas constriction of blood vessels results in increases in bloodpressure. The blood vessels are controlled through local neuralsignaling that is largely under parasympathetic control, and circulatinghormones that are largely under sympathetic control, as well as othercirculating proteins. Blood pressure increases following stimulation ofthe angiotensin receptors, which results in vasoconstriction.Angiotensin receptors are stimulated by angiotensin II, which isconverted from angiotensin I through the angiotensin converting enzyme(ACE). Angiotensin II is a potent vasoconstrictor and actively inhibitsbradykinin, which is a potent vasodilator.

Therefore, angiotensin converting enzyme is a common target of bloodpressure therapy. ACE inhibitors such as lisinopril promote vasodilationwhich, ultimately, reduces the bioavailability of angiotensin-II.Similarly, angiotensin II receptor antagonists such as losartan act ascompetitive inhibitors, which decrease the number of receptors that areavailable to bind to angiotensin-II. Despite the method used forpromoting vasodilation (through reductions in ACE or receptorinhibition) the end result, on average in the population, isvasodilation which results in a drop in blood pressure due to theinverse relationship between the size of the vessel and the pressureexerted on the vessel, all else being equal. Despite this benefit thereis a “bell-curve” response to these therapies in humans. Not allindividuals are responsive to vasodilator therapy.

Several functional polymorphisms of the genes that encode for ACE andangiotensin-II receptors exist, which can affect how a subject respondsto vasodilation.

Examples of functional ACE polymorphisms include the insertion ordeletion polymorphisms such as a 287 base pair fragment (Ulgen et al.,Coron Artery Dis 18:153-157 (2007)). The rs1799752 polymorphism is aninsertion/deletion in an intron of the ACE gene, and with the sequence(SEQ ID NO:12) shown below, where sequences in the bracket are theinsertion/deletion.

TCCCATTTCTCTAGACCTGCTGCCT[-/ATACAGTCACTTTTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTCGCCC]ATACAGTCACTTTTATGTG GTTTCG.The deletion removes the bracketed nucleic acid segment so that thers1799752 polymorphism will have the following sequence (SEQ ID NO:35).

TCCCATTTCTCTAGACCTGCTGCCTATACAGTCACTTTTATGTGGTTTCG.

Research indicates that such an ACE deletion polymorphism results inhigher ACE plasma levels and greater reduction in ejection fraction inpatients following myocardial infraction (likely from elevations inblood pressure) (McNamara et al., J Am Coll Cardiol 44:2019-2026 (2004);Pilati et al., Congest Heart Fail 10:87-93 (2004). In addition, patientswith the deletion polymorphism are more likely to have left-ventricularhypertrophy when compared to patients with the insertion polymorphism(left-ventricular hypertrophy results secondary to prolonged exposure tohigh blood pressure). Subjects with the deletion polymorphism wouldtherefore be most responsive to ACE-inhibition or angiotensin-IIreceptor inhibition.

At least one functional variant of angiotensin has been found in humans:a cytosine to threonine substitution at nucleotide 4072 (Pilbrow et al.,Hypertension 49:322-327 (2007); Tang et al., Am Heart J 143:854-860(2002)). Human angiotensinogen is expressed from the AGT gene. A cDNAnucleotide sequence for human angiotensinogen is provided below as SEQID NO:13 (accession number NM_000029.3 GI:188595658, from the NCBIdatabase).

   1 ATCCCATGAG CGGGCAGCAG GGTCAGAAGT GGCCCCCGTG   41TTGCCTAAGC AAGACTCTCC CCTGCCCTCT GCCCTCTGCA   81CCTCCGGCCT GCATGTCCCT GTGGCCTCTT GGGGGTACAT  121CTCCCGGGGC TGGGTCAGAA GGCCTGGGTG GTTGGCCTCA  161GGCTGTCACA CACCTAGGGA GATGCTCCCG TTTCTGGGAA  201CCTTGGCCCC GACTCCTGCA AACTTCGGTA AATGTGTAAC  241TCGACCCTGC ACCGGCTCAC TCTGTTCAGC AGTGAAACTC  281TGCATCGATC ACTAAGACTT CCTGGAAGAG GTCCCAGCGT  321GAGTGTCGCT TCTGGCATCT GTCCTTCTGG CCAGCCTGTG  361GTCTGGCCAA GTGATGTAAC CCTCCTCTCC AGCCTGTGCA  401CAGGCAGCCT GGGAACAGCT CCATCCCCAC CCCTCAGCTA  441TAAATAGGGC ATCGTGACCC GGCCGGGGGA AGAAGCTGCC  481GTTGTTCTGG GTACTACAGC AGAAGGGTAT GCGGAAGCGA  521GCACCCCAGT CTGAGATGGC TCCTGCCGGT GTGAGCCTGA  561GGGCCACCAT CCTCTGCCTC CTGGCCTGGG CTGGCCTGGC  601TGCAGGTGAC CGGGTGTACA TACACCCCTT CCACCTCGTC  641ATCCACAATG AGAGTACCTG TGAGCAGCTG GCAAAGGCCA  681ATGCCGGGAA GCCCAAAGAC CCCACCTTCA TACCTGCTCC  721AATTCAGGCC AAGACATCCC CTGTGGATGA AAAGGCCCTA  761CAGGACCAGC TGGTGCTAGT CGCTGCAAAA CTTGACACCG  801AAGACAAGTT GAGGGCCGCA ATGGTCGGGA TGCTGGCCAA  841CTTCTTGGGC TTCCGTATAT ATGGCATGCA CAGTGAGCTA  881TGGGGCGTGG TCCATGGGGC CACCGTCCTC TCCCCAACGG  921CTGTCTTTGG CACCCTGGCC TCTCTCTATC TGGGAGCCTT  961GGACCACACA GCTGACAGGC TACAGGCAAT CCTGGGTGTT 1001CCTTGGAAGG ACAAGAACTG CACCTCCCGG CTGGATGCGC 1041ACAAGGTCCT GTCTGCCCTG CAGGCTGTAC AGGGCCTGCT 1081AGTGGCCCAG GGCAGGGCTG ATAGCCAGGC CCAGCTGCTG 1121CTGTCCACGG TGGTGGGCGT GTTCACAGCC CCAGGCCTGC 1161ACCTGAAGCA GCCGTTTGTG CAGGGCCTGG CTCTCTATAC 1201CCCTGTGGTC CTCCCACGCT CTCTGGACTT CACAGAACTG 1241GATGTTGCTG CTGAGAAGAT TGACAGGTTC ATGCAGGCTG 1281TGACAGGATG GAAGACTGGC TGCTCCCTGA  T GGGAGCCAG 1321TGTGGACAGC ACCCTGGCTT TCAACACCTA CGTCCACTTC 1361CAAGGGAAGA TGAAGGGCTT CTCCCTGCTG GCCGAGCCCC 1401AGGAGTTCTG GGTGGACAAC AGCACCTCAG TGTCTGTTCC 1441CATGCTCTCT GGCATGGGCA CCTTCCAGCA CTGGAGTGAC 1481ATCCAGGACA ACTTCTCGGT GACTCAAGTG CCCTTCACTG 1521AGAGCGCCTG CCTGCTGCTG ATCCAGCCTC ACTATGCCTC 1561TGACCTGGAC AAGGTGGAGG GTCTCACTTT CCAGCAAAAC 1601TCCCTCAACT GGATGAAGAA ACTATCTCCC CGGACCATCC 1641ACCTGACCAT GCCCCAACTG GTGCTGCAAG GATCTTATGA 1681CCTGCAGGAC CTGCTCGCCC AGGCTGAGCT GCCCGCCATT 1721CTGCACACCG AGCTGAACCT GCAAAAATTG AGCAATGACC 1761GCATCAGGGT GGGGGAGGTG CTGAACAGCA TTTTTTTTGA 1801GCTTGAAGCG GATGAGAGAG AGCCCACAGA GTCTACCCAA 1841CAGCTTAACA AGCCTGAGGT CTTGGAGGTG ACCCTGAACC 1881GCCCATTCCT GTTTGCTGTG TATGATCAAA GCGCCACTGC 1921CCTGCACTTC CTGGGCCGCG TGGCCAACCC GCTGAGCACA 1961GCATGAGGCC AGGGCCCCAG AACACAGTGC CTGGCAAGGC 2001CTCTGCCCCT GGCCTTTGAG GCAAAGGCCA GCAGCAGATA 2041ACAACCCCGG ACAAATCAGC GATGTGTCAC CCCCAGTCTC 2081CCACCTTTTC TTCTAATGAG TCGACTTTGA GCTGGAAAGC 2121AGCCGTTTCT CCTTGGTCTA AGTGTGCTGC ATGGAGTGAG 2161CAGTAGAAGC CTGCAGCGGC ACAAATGCAC CTCCCAGTTT 2201GCTGGGTTTA TTTTAGAGAA TGGGGGTGGG GAGGCAAGAA 2241CCAGTGTTTA GCGCGGGACT ACTGTTCCAA AAAGAATTCC 2281AACCGACCAG CTTGTTTGTG AAACAAAAAA GTGTTCCCTT 2321TTCAAGTTGA GAACAAAAAT TGGGTTTTAA AATTAAAGTA 2361TACATTTTTG CATTGCCTTC GGTTTGTATT TAGTGTCTTG 2401AATGTAAGAA CATGACCTCC GTGTAGTGTC TGTAATACCT 2441TAGTTTTTTC CACAGATGCT TGTGATTTTT GAACAATACG 2481TGAAAGATGC AAGCACCTGA ATTTCTGTTT GAATGCGGAA 2521CCATAGCTGG TTATTTCTCC CTTGTGTTAG TAATAAACGT 2561 CTTGCCACAA TAAGCCTCCA2581 AAAAAAA

The rs699 single nucleotide polymorphism (SNP) is present in the AGTgene, where the variable nucleotide is at about position 1311 in SEQ IDNO:13 (underlined), which can be in thymine some individuals andcytosine in others. The rs699 sequence (SEQ ID NO:14) is shown below,where the underlined C/T is the SNP.

GGATGGAAGACTGGCTGCTCCCTGA[C/T]GGGAGCCAGTGTGGACAG CACCCTG.

The human angiotensinogen polypeptide encoded by the AGT cDNA with SEQID NO:13 has the following sequence (SEQ ID NO:15).

  1 MRKRAPQSEM APAGVSLRAT ILCLLAWAGL AAGDRVYIHP  41FHLVIHNEST CEQLAKANAG KPKDPTFIPA PIQAKTSPVD  81EKALQDQLVL VAAKLDTEDK LRAAMVGMLA NFLGFRIYGM 121HSELWGVVHG ATVLSPTAVF GTLASLYLGA LDHTADRLQA 161ILGVPWKDKN CTSRLDAHKV LSALQAVQGL LVAQGRADSQ 201AQLLLSTVVG VFTAPGLHLK QPFVQGLALY TPVVLPRSLD 241FTELDVAAEK IDRFMQAVTG WKTGCSL M GA SVDSTLAFNT 281YVHFQGKMKG FSLLAEPQEF WVDNSTSVSV PMLSGMGTFQ 321HWSDIQDNFS VTQVPFTESA CLLLIQPHYA SDLDKVEGLT 361FQQNSLNWMK KLSPRTIHLT MPQLVLQGSY DLQDLLAQAE 401LPAILHTELN LQKLSNDRIR VGEVLNSIFF ELEADEREPT 441ESTQQLNKPE VLEVTLNRPF LFAVYDQSAT ALHFLGRVAN 481 PLSTA

Note that the underlined methionine at position 268 of SEQ ID NO:15 ismethionine because some individuals have nucleotide sequence SEQ IDNO:13, where the nucleotide at about position 1311 is thymine. However,position 268 of SEQ ID NO:15 can be threonine in some individualsbecause those individuals have cytosine at nucleotide position 1311 inSEQ ID NO:13.

The threonine polymorphism of angiotensin results in higher angiotensinlevels and higher resting blood pressure values. Therefore, patientswith the threonine genetic variant will benefit primarily from an ACEinhibitor (preventing the conversion of the higher levels of angiotensinI to angiotensin II) or an angiotensin receptor inhibitor.

An example of a functional polymorphism of an angiotensin II receptortype-1 involves an adenine to cytosine substitution at nucleotide 1166(Miller et al. Kidney Int 56:2173-2180 (1999); Baudin, Pharmacogenomics3:65-73 (2002)). Human angiotensin II receptor type-1 is expressed fromthe AGT1R gene. One example of an AGT1R single nucleotide polymorphismis the so-called A1166→C polymorphism, which is in the 3′ untranslatedregion of the AGT1R gene. This A1166→C polymorphism is also identifiedas the rs5186 single nucleotide polymorphism (SNP), which has thefollowing sequence (SEQ ID NO:16) where the underlined A/C is thevariable SNP site.

TGCAGCACTTCACTACCAAATGAGC[A/C]TTAGCTACTTTTCAGAATTG AAGGA.A portion of a 3′ untranslated region of the AGT1R gene with NCBIaccession number NG_008468.1 (GI:198041751) is shown below (SEQ IDNO:17) that contains the rs5186 SNP with the variant nucleotide(adenine) identified below in bold and with underlining.

48961 ATTCAACTAG GCATCATACG TGACTGTAGA ATTGCAGATA 49001TTGTGGACAC GGCCATGCCT ATCACCATTT GTATAGCTTA 49041TTTTAACAAT TGCCTGAATC CTCTTTTTTA TGGCTTTCTG 49081GGGAAAAAAT TTAAAAGATA TTTTCTCCAG CTTCTAAAAT 49121ATATTCCCCC AAAAGCCAAA TCCCACTCAA ACCTTTCAAC 49181AAAAATGAGC ACGCTTTCCT ACCGCCCCTC AGATAATGTA 49201AGCTCATCCA CCAAGAAGCC TGCACCATGT TTTGAGGTTG 49241AGTGACATGT TCGAAACCTG TCCATAAAGT AATTTTGTGA 49301AAGAAGGAGC AAGAGAACAT TCCTCTGCAG CACTTCACTA 49321 CCAAATGAGC  ATTAGCTACT TTTCAGAATT GAAGGAGAAA 49361ATGCATTATG TGGACTGAAC CGACTTTTCT AAAGCTCTGA 49401ACAAAAGCTT TTCTTTCCTT TTGCAACAAG ACAAAGCAAA 49441GCCACATTTT GCATTAGACA GATGACGGCT GCTCGAAGAA 49481CAATGTCAGA AACTCGATGA ATGTGTTGAT TTGAGAAATT 49521TTACTGACAG AAATGCAATC TCCCTAGCCT GCTTTTGTCC 49561TGTTATTTTT TATTTCCACA TAAAGGTATT TAGAATATAT 49601TAAATCGTTA GAGGAGCAAC AGGAGATGAG AGTTCCAGAT 49641TGTTCTGTCC AGTTTCCAAA GGGCAGTAAA GTTTTCGTGC

This polymorphism has been shown to influence resting blood pressurevalues which suggest which patients may benefit more from angiotensin-IIreceptor inhibition. Specifically, patients with the C variant of theangiotensin receptor type I tend to demonstrate higher resting bloodpressure values, have more detrimental cardiovascular events, and have agreater chance of developing high blood pressure during pregnancy, whencompared to the A variant. Subjects with the C variant will therefore bemore responsive to angiotensin receptor blockers.

A cDNA sequence for human angiotensin II receptor is provided in theNCBI database as accession number X65699.1 (GI:510983), which has thefollowing sequence (SEQ ID NO:18).

   1 GGCAGCAGCG AGTGACAGGA CGTCTGGACC GGCGCGCCGC   41TAGCAGCTCT GCCGGGCCGC GGCGGTGATC GATGGGAGCG   81GCTGGAGCGG ACCCAGCGAG TGAGGGCGCA CAGCCGGACG  121CCGAGGCGGC GGGCGGGAGA CCGCACCGCG ACGCCGGCCC  161TCGGCGGACG AGTCGAGCGC CCGGGCGCGG GTGTATTTGA  201TATAGTGTTT GCAACAAATT CGACCCAGGT GATCAAAATG  241ATTCTCAACT CTTCTACTGA AGATGGTATT AAAAGAATCC  281AAGATGATTG TCCCAAAGCT GGAAGGCATA ATTACATATT  321TGTCATGATT CCTACTTTAT ACAGTATCAT CTTTGTGGTG  361GGAATATTTG GAAACAGCTT GGTGGTGATA GTCATTTACT  401TTTATATGAA GCTGAAGACT GTGGCCAGTG TTTTTCTTTT  441GAATTTAGCA CTGGCTGACT TATGCTTTTT ACTGACTTTG  481CCACTATGGG CTGTCTACAC AGCTATGGAA TACCGCTGGC  521CCTTTGGCAA TTACCTATGT AAGATTGCTT CAGCCAGCGT  561CAGTTTCAAC CTGTACGCTA GTGTGTTTCT ACTCACGTGT  601CTCAGCATTG ATCGATACCT GGCTATTGTT CACCCAATGA  641AGTCCCGCCT TCGACGCACA ATGCTTGTAG CCAAAGTCAC  681CTGCATCATC ATTTGGCTGC TGGCAGGCTT GGCCAGTTTG  721CCAGCTATAA TCCATCGAAA TGTATTTTTC ATTGAGAACA  761CCAATATTAC AGTTTGTGCT TTCCATTATG AGTCCCAAAA  801TTCAACCCTC CCGATAGGGC TGGGCCTGAC CAAAAATATA  841CTGGGTTTCC TGTTTCCTTT TCTGATCATT CTTACAAGTT  881ATACTCTTAT TTGGAAGGCC CTAAAGAAGG CTTATGAAAT  921TCAGAAGAAC AAACCAAGAA ATGATGATAT TTTTAAGATA  961ATTATGGCAA TTGTGCTTTT CTTTTTCTTT TCCTGGATTC 1001CCCACCAAAT ATTCACTTTT CTGGATGTAT TGATTCAACT 1041AGGCATCATA CGTGACTGTA GAATTGCAGA TATTGTGGAC 1081ACGGCCATGC CTATCACCAT TTGTATAGCT TATTTTAACA 1121ATTGCCTGAA TCCTCTTTTT TATGGCTTTC TGGGGAAAAA 1161ATTTAAAAGA TATTTTCTCC AGCTTCTAAA ATATATTCCC 1201CCAAAAGCCA AATCCCACTC AAACCTTTCA ACAAAAATGA 1241GCACGCTTTC CTACCGCCCC TCAGATAATG TAAGCTCATC 1281CACCAAGAAG CCTGCACCAT GTTTTGAGGT TGAGTGACAT 1321GTTCGAAACC TGTCCATAAA GTAATTTTGT GAAAGAAGGA 1361GCAAGAGAAC ATTCCTCTGC AGCACTTCAC TACCAAATGA 1401 GC ATTAGCTA CTTTTCAGAA TTGAAGGAGA AAATGCATTA 1441TGTGGACTGA ACCGACTTTT CTAAAGCTCT GAACAAAAGC 1481TTTTCTTTCC TTTTGCAACA AGACAAAGCA AAGCCACATT 1521TTGCATTAGA CAGATGACGG CTGCTCGAAG AACAATGTCA 1561GAAACTCGAT GAATGTGTTG ATTTGAGAAA TTTTACTGAC 1601AGAAATGCAA TCTCCCTAGC CTGCTTTTGT CCTGTTATTT 1641TTTATTTCCA CATAAAGGTA TTTAGAATAT ATTAACTCGT 1681TAGAGGAGCA ACAGGAGATG AGAGTTCCAG ATTGTTCTGT 1721CCAGTTTCCA AAGGGCAGTA AAGTTTTCGT GCCTGTTTTC 1761AGCTATTAGC AACTGTGCCT ACACTTGCAC CTGGTCTGCA 1801CATTTTGTAC AAAGATATGC TTAAGCAGTA GTCGTCAAGT 1841TGCAGATCTT TGTTGTGAAA TTCAACCTGT GTCTTATAGG 1881TTTACACTGC CAAAACAATG CCCGTAAGAT GGCTTATTTG 1921TATAATGGTG TTACCTAAAG TCACATATAA AAGTTAAACT 1961ACTTGTAAAG GTGCTGCACT GGTCCCAAGT AGTAGTGTCT 2001TCCTAGTATA TTAGTTTGAT TTAATATCTG AGAAGTGTAT 2041ATAGTTTGTG GTAAAAAGAT TATATATCAT AAAGTATGCC 2081TTCCTGTTTA AAAAAAGTAT ATATTCTACA CATATATGTA 2121TATGTATATC TATATCTCTA AACTGCTGTT AATTGATTAA 2161AATCTGGCAA AGTTATATTT ACCCC

In addition to angiotensin, angiotensin II receptors and ACE, renin hasbeen shown to be a potent vasoconstrictor that can result in high bloodpressure. Renin converts angiotensinogen to angiotensin I which canresult in vasoconstriction due to the down-stream effects (angiotensin-Iconversion to angiotensin II through ACE). One example of a functionaland common renin polymorphism (Vangjeli et al., CirculationCardiovascular genetics 3:53-59 (2010)) can influence the blood pressureresponse to vasodilator therapy. This renin polymorphism is present inrs12750834. The nucleotide sequence surrounding the renin rs12750834single nucleotide polymorphism is shown below, where the underlined A/Gin the sequence (SEQ ID NO:19) is the SNP.

AGAACACCAAAGCAGGCTTAATCTG[A/G]GGGCACTTACAGAGACTG CTTTAAA.The complementary sequence of SEQ ID NO:19 is the following sequence(SEQ ID NO:20).

TTTAAAGCAGTCTCTGTAAGTGCCC[C/T]CAGATTAAGCCTGCTTTG GTGTTCT.Note that the cytosine to thymine substitution is a guanine to adeninesubstitution in the opposite strand.

The rs12750834 SNP contains a cytosine to thymine substitution, or aguanine to adenine substitution depending upon the DNA strand, at aboutnucleotide position 5312 upstream of the renin start site. The cytosine(guanine) variant of renin has been shown to correlate with greaterreduction in blood pressure upon administration of angiotensin IIreceptor blockers such as valsartan.

Sodium/Diuretic Regulation of Blood Pressure

The kidneys are the center of long-term blood pressure regulation.Alterations in Na⁺ reabsorption in the kidneys result in alterations influid retention, which leads to increases or decreases in blood plasmavolume as well as to changes in the pressure against the vessels. Thereare several proteins that are important in renal Na⁺ handling and in theresponse to diuretic therapy including the epithelial Na⁺ channels,alpha-adducin, the Na⁺Cl⁻ co-transporter, and lysine deficient proteinkinase-1 (WNK).

The epithelial sodium (Na⁺) channel is responsible for Na⁺ reabsorptionon the apical portion of epithelial cells in the kidneys. The Na⁺channel is made up of three different subunits: the alpha, beta, andgamma. The alpha subunit of the epithelial Na⁺ channel is highlyfunctional and removal of this subunit abolishes channel activity incell and animal models. The gamma subunit is also extremely important inchannel function. Functional gamma genetic variants result inpseudohypoaldosteronism type-I and Liddle's syndrome, two severe geneticdiseases resulting in salt wasting and high salt conservation (saltsensitivity), respectively. Adducin is made up of an alpha, beta, andgamma subunit. The alpha subunit increases sodium (Na⁺) reabsorption inthe kidneys through the activity of Na⁺K⁺ ATPase (which moves Na⁺ andpotassium into and out of cells). The sodium (Na⁺) chloride (Cl⁻)co-transporter is important in regulating Na⁺ and Cl⁻ movement betweenthe kidney and the rest of the body. Active Na⁺—Cl⁻ transport results inNa⁺ reabsorption and can, therefore, result in higher blood pressure.The WNK1 protein is a key regulator of long-term Na⁺ and chloride Cl⁻reabsorption in the kidneys. WNK1 regulates the activity of Na⁺—Cl⁻co-transporters. If a patient has a more active WNK1 genotype, theylikely have greater Na⁺ and Cl⁻ reabsorption in the kidneys which canincrease blood volume and, therefore, pressure on the vessels.

A functional and common polymorphism of the gene that encodes theepithelial Na+channel (SCNN1A) has been identified, where thepolymorphism is an alanine to threonine substitution at about position663-722. A cDNA sequence for the human SCNN1A gene is available from theNCBI database as accession number NM_001159576.1 (GI:227430288). Thissequence is provided below as SEQ ID NO:21.

   1 AAACAGAAGG CAGATAGAGA GGGAGTGAGA GGCAGGAGCT   41GAGACACAGA TCCTGGAGGA AGAAGACCAA AGGAAGGGGG   81CAGAGACAGA AAGGGAGGTG CTAGGACAAA ACTCGAAAGG  121TGGCCCTATC AGGGAAGCAG AGGAGAGGCC GTTCTAGGGA  161AGCCCAGCTC CGGCACTTTT GGCCCCAACT CCCGCAGGTC  201TGCTGGCTCC AGGAAAGGTG GAGGAGGGAG GGAGGAGTGG  241GAGAATGTGG GCGCAGGGTG GGACATGGGC ATGGCCAGGG  281GCAGCCTCAC TCGGGTTCCA GGGGTGATGG GAGAGGGCAC  321TCAGGGCCCA GAGCTCAGCC TTGACCCTGA CCCTTGCTCT  361CCCCAATCCA CTCCGGGGCT CATGAAGGGG AACAAGCTGG  401AGGAGCAGGA CCCTAGACCT CTGCAGCCCA TACCAGGTCT  441CATGGAGGGG AACAAGCTGG AGGAGCAGGA CTCTAGCCCT  481CCACAGTCCA CTCCAGGGCT CATGAAGGGG AACAAGCGTG  521AGGAGCAGGG GCTGGGCCCC GAACCTGCGG CGCCCCAGCA  561GCCCACGGCG GAGGAGGAGG CCCTGATCGA GTTCCACCGC  601TCCTACCGAG AGCTCTTCGA GTTCTTCTGC AACAACACCA  641CCATCCACGG CGCCATCCGC CTGGTGTGCT CCCAGCACAA  681CCGCATGAAG ACGGCCTTCT GGGCAGTGCT GTGGCTCTGC  721ACCTTTGGCA TGATGTACTG GCAATTCGGC CTGCTTTTCG  761GAGAGTACTT CAGCTACCCC GTCAGCCTCA ACATCAACCT  801CAACTCGGAC AAGCTCGTCT TCCCCGCAGT GACCATCTGC  841ACCCTCAATC CCTACAGGTA CCCGGAAATT AAAGAGGAGC  881TGGAGGAGCT GGACCGCATC ACAGAGCAGA CGCTCTTTGA  921CCTGTACAAA TACAGCTCCT TCACCACTCT CGTGGCCGGC  961TCCCGCAGCC GTCGCGACCT GCGGGGGACT CTGCCGCACC 1001CCTTGCAGCG CCTGAGGGTC CCGCCCCCGC CTCACGGGGC 1041CCGTCGAGCC CGTAGCGTGG CCTCCAGCTT GCGGGACAAC 1081AACCCCCAGG TGGACTGGAA GGACTGGAAG ATCGGCTTCC 1121AGCTGTGCAA CCAGAACAAA TCGGACTGCT TCTACCAGAC 1161ATACTCATCA GGGGTGGATG CGGTGAGGGA GTGGTACCGC 1201TTCCACTACA TCAACATCCT GTCGAGGCTG CCAGAGACTC 1241TGCCATCCCT GGAGGAGGAC ACGCTGGGCA ACTTCATCTT 1281CGCCTGCCGC TTCAACCAGG TCTCCTGCAA CCAGGCGAAT 1321TACTCTCACT TCCACCACCC GATGTATGGA AACTGCTATA 1361CTTTCAATGA CAAGAACAAC TCCAACCTCT GGATGTCTTC 1401CATGCCTGGA ATCAACAACG GTCTGTCCCT GATGCTGCGC 1441GCAGAGCAGA ATGACTTCAT TCCCCTGCTG TCCACAGTGA 1481CTGGGGCCCG GGTAATGGTG CACGGGCAGG ATGAACCTGC 1521CTTTATGGAT GATGGTGGCT TTAACTTGCG GCCTGGCGTG 1561GAGACCTCCA TCAGCATGAG GAAGGAAACC CTGGACAGAC 1601TTGGGGGCGA TTATGGCGAC TGCACCAAGA ATGGCAGTGA 1641TGTTCCTGTT GAGAACCTTT ACCCTTCAAA GTACACACAG 1681CAGGTGTGTA TTCACTCCTG CTTCCAGGAG AGCATGATCA 1721AGGAGTGTGG CTGTGCCTAC ATCTTCTATC CGCGGCCCCA 1761GAACGTGGAG TACTGTGACT ACAGAAAGCA CAGTTCCTGG 1801GGGTACTGCT ACTATAAGCT CCAGGTTGAC TTCTCCTCAG 1841ACCACCTGGG CTGTTTCACC AAGTGCCGGA AGCCATGCAG 1881CGTGACCAGC TACCAGCTCT CTGCTGGTTA CTCACGATGG 1921CCCTCGGTGA CATCCCAGGA ATGGGTCTTC CAGATGCTAT 1961CGCGACAGAA CAATTACACC GTCAACAACA AGAGAAATGG 2001AGTGGCCAAA GTCAACATCT TCTTCAAGGA GCTGAACTAC 2041AAAACCAATT CTGAGTCTCC CTCTGTCACG ATGGTCACCC 2081TCCTGTCCAA CCTGGGCAGC CAGTGGAGCC TGTGGTTCGG 2121CTCCTCGGTG TTGTCTGTGG TGGAGATGGC TGAGCTCGTC 2161TTTGACCTGC TGGTCATCAT GTTCCTCATG CTGCTCCGAA 2201GGTTCCGAAG CCGATACTGG TCTCCAGGCC GAGGGGGCAG 2241GGGTGCTCAG GAGGTAGCCT CCACCCTGGC ATCCTCCCCT 2281CCTTCCCACT TCTGCCCCCA CCCCATGTCT CTGTCCTTGT 2321CCCAGCCAGG CCCTGCTCCC TCTCCAGCCT TGACAGCCCC 2361TCCCCCTGCC TATGCCACCC TGGGCCCCCG CCCATCTCCA 2401GGGGGCTCTG CAGGGGCCAG TTCCTCC A CC TGTCCTCTGG 2441GGGGGCCCTG AGAGGGAAGG AGAGGTTTCT CACACCAAGG 2481CAGATGCTCC TCTGGTGGGA GGGTGCTGGC CCTGGCAAGA 2521TTGAAGGATG TGCAGGGCTT CCTCTCAGAG CCGCCCAAAC 2561TGCCGTTGAT GTGTGGAGGG GAAGCAAGAT GGGTAAGGGC 2601TCAGGAAGTT GCTCCAAGAA CAGTAGCTGA TGAAGCTGCC 2641CAGAAGTGCC TTGGCTCCAG CCCTGTACCC CTTGGTACTG 2681CCTCTGAACA CTCTGGTTTC CCCACCCAAC TGCGGCTAAG 2721TCTCTTTTTC CCTTGGATCA GCCAAGCGAA ACTTGGAGCT 2761TTGACAAGGA ACTTTCCTAA GAAACCGCTG ATAACCAGGA 2801CAAAACACAA CCAAGGGTAC ACGCAGGCAT GCACGGGTTT 2841CCTGCCCAGC GACGGCTTAA GCCAGCCCCC GACTGGCCTG 2881GCCACACTGC TCTCCAGTAG CACAGATGTC TGCTCCTCCT 2921CTTGAACTTG GGTGGGAAAC CCCACCCAAA AGCCCCCTTT 2961GTTACTTAGG CAATTCCCCT TCCCTGACTC CCGAGGGCTA 3001GGGCTAGAGC AGACCCGGGT AAGTAAAGGC AGACCCAGGG 3041CTCCTCTAGC CTCATACCCG TGCCCTCACA GAGCCATGCC 3081CCGGCACCTC TGCCCTGTGT CTTTCATACC TCTACATGTC 3121TGCTTGAGAT ATTTCCTCAG CCTGAAAGTT TCCCCAACCA 3161TCTGCCAGAG AACTCCTATG CATCCCTTAG AACCCTGCTC 3201AGACACCATT ACTTTTGTGA ACGCTTCTGC CACATCTTGT 3241CTTCCCCAAA ATTGATCACT CCGCCTTCTC CTGGGCTCCC 3281GTAGCACACT ATAACATCTG CTGGAGTGTT GCTGTTGCAC 3321CATACTTTCT TGTACATTTG TGTCTCCCTT CCCAACTAGA 3361CTGTAAGTGC CTTGCGGTCA GGGACTGAAT CTTGCCCGTT 3401TATGTATGCT CCATGTCTAG CCCATCATCC TGCTTGGAGC 3441AAGTAGGCAG GAGCTCAATA AATGTTTGTT GCATGAAGGA 3481 AAAAAAAAAA AAAAAAA

The rs2228576 single nucleotide polymorphism (SNP) is present in theSCNN1A gene, where the variable nucleotide is at about position 2428 inSEQ ID NO:21 (underlined), which can be adenine in some individuals andguanine in others. The rs2228576 sequence (SEQ ID NO:22) is shown below,where the underlined A/G is the SNP.

GGGCTCTGCAGGGGCCAGTTCCTCC[A/G]CCTGTCCTCTGGGGGGGCC CTGAGA

The human the epithelial Na+channel encoded by the SCNN1A cDNA with SEQID NO:21 has the following sequence (SEQ ID NO:23).

  1 MGMARGSLTR VPGVMGEGTQ GPELSLDPDP CSPQSTPGLM  41KGNKLEEQDP RPLQPIPGLM EGNKLEEQDS SPPQSTPGLM  81KGNKREEQGL GPEPAAPQQP TAEEEALIEF HRSYRELFEF 121FCNNTTIHGA IRLVCSQHNR MKTAFWAVLW LCTFGMMYWQ 161FGLLFGEYFS YPVSLNINLN SDKLVFPAVT ICTLNPYRYP 201EIKEELEELD RITEQTLFDL YKYSSFTTLV AGSRSRRDLR 241GTLPHPLQRL RVPPPPHGAR RARSVASSLR DNNPQVDWKD 281WKIGFQLCNQ NKSDCFYQTY SSGVDAVREW YRFHYINILS 321RLPETLPSLE EDTLGNFIFA CRFNQVSCNQ ANYSHFHHPM 361YGNCYTFNDK NNSNLWMSSM PGINNGLSLM LRAEQNDFIP 401LLSTVTGARV MVHGQDEPAF MDDGGFNLRP GVETSISMRK 441ETLDRLGGDY GDCTKNGSDV PVENLYPSKY TQQVCIHSCF 481QESMIKECGC AYIFYPRPQN VEYCDYRKHS SWGYCYYKLQ 521VDFSSDHLGC FTKCRKPCSV TSYQLSAGYS RWPSVTSQEW 561VFQMLSRQNN YTVNNKRNGV AKVNIFFKEL NYKTNSESPS 601VTMVTLLSNL GSQWSLWFGS SVLSVVEMAE LVFDLLVIMF 641LMLLRRFRSR YWSPGRGGRG AQEVASTLAS SPPSHFCPHP 681MSLSLSQPGP APSPALTAPP PAYATLGPRP SPGGSAGASS 721 S T CPLGGP

Another cDNA sequence for the human SCNN1A gene with the same SNP isavailable from the NCBI database as accession number NM_001038.5(GI:227430285). This sequence is provided below as SEQ ID NO:24.

   1 CTTGCCTGTC TGCGTCTAAA GCCCCTGCCC AGAGTCCGCC   41TTCTCAGGTC CAGTACTCCC AGTTCACCTG CCCTCGGGAG   81CCCTCCTTCC TTCGGAAAAC TCCCGGCTCT GACTCCTCCT  121CAGCCCCTCC CCCCGCCCTG CTCACCTTTA ATTGAGATGC  161TAATGAGATT CCTGTCGCTT CCATCCCTGG CCGGCCAGCG  201GGCGGGCTCC CCAGCCAGGC CGCTGCACCT GTCAGGGGAA  241CAAGCTGGAG GAGCAGGACC CTAGACCTCT GCAGCCCATA  281CCAGGTCTCA TGGAGGGGAA CAAGCTGGAG GAGCAGGACT  321CTAGCCCTCC ACAGTCCACT CCAGGGCTCA TGAAGGGGAA  361CAAGCGTGAG GAGCAGGGGC TGGGCCCCGA ACCTGCGGCG  401CCCCAGCAGC CCACGGCGGA GGAGGAGGCC CTGATCGAGT  441TCCACCGCTC CTACCGAGAG CTCTTCGAGT TCTTCTGCAA  481CAACACCACC ATCCACGGCG CCATCCGCCT GGTGTGCTCC  521CAGCACAACC GCATGAAGAC GGCCTTCTGG GCAGTGCTGT  561GGCTCTGCAC CTTTGGCATG ATGTACTGGC AATTCGGCCT  601GCTTTTCGGA GAGTACTTCA GCTACCCCGT CAGCCTCAAC  641ATCAACCTCA ACTCGGACAA GCTCGTCTTC CCCGCAGTGA  681CCATCTGCAC CCTCAATCCC TACAGGTACC CGGAAATTAA  721AGAGGAGCTG GAGGAGCTGG ACCGCATCAC AGAGCAGACG  761CTCTTTGACC TGTACAAATA CAGCTCCTTC ACCACTCTCG  801TGGCCGGCTC CCGCAGCCGT CGCGACCTGC GGGGGACTCT  841GCCGCACCCC TTGCAGCGCC TGAGGGTCCC GCCCCCGCCT  881CACGGGGCCC GTCGAGCCCG TAGCGTGGCC TCCAGCTTGC  921GGGACAACAA CCCCCAGGTG GACTGGAAGG ACTGGAAGAT  961CGGCTTCCAG CTGTGCAACC AGAACAAATC GGACTGCTTC 1001TACCAGACAT ACTCATCAGG GGTGGATGCG GTGAGGGAGT 1041GGTACCGCTT CCACTACATC AACATCCTGT CGAGGCTGCC 1081AGAGACTCTG CCATCCCTGG AGGAGGACAC GCTGGGCAAC 1121TTCATCTTCG CCTGCCGCTT CAACCAGGTC TCCTGCAACC 1161AGGCGAATTA CTCTCACTTC CACCACCCGA TGTATGGAAA 1201CTGCTATACT TTCAATGACA AGAACAACTC CAACCTCTGG 1241ATGTCTTCCA TGCCTGGAAT CAACAACGGT CTGTCCCTGA 1281TGCTGCGCGC AGAGCAGAAT GACTTCATTC CCCTGCTGTC 1321CACAGTGACT GGGGCCCGGG TAATGGTGCA CGGGCAGGAT 1361GAACCTGCCT TTATGGATGA TGGTGGCTTT AACTTGCGGC 1401CTGGCGTGGA GACCTCCATC AGCATGAGGA AGGAAACCCT 1441GGACAGACTT GGGGGCGATT ATGGCGACTG CACCAAGAAT 1481GGCAGTGATG TTCCTGTTGA GAACCTTTAC CCTTCAAAGT 1521ACACACAGCA GGTGTGTATT CACTCCTGCT TCCAGGAGAG 1561CATGATCAAG GAGTGTGGCT GTGCCTACAT CTTCTATCCG 1601CGGCCCCAGA ACGTGGAGTA CTGTGACTAC AGAAAGCACA 1641GTTCCTGGGG GTACTGCTAC TATAAGCTCC AGGTTGACTT 1681CTCCTCAGAC CACCTGGGCT GTTTCACCAA GTGCCGGAAG 1721CCATGCAGCG TGACCAGCTA CCAGCTCTCT GCTGGTTACT 1761CACGATGGCC CTCGGTGACA TCCCAGGAAT GGGTCTTCCA 1801GATGCTATCG CGACAGAACA ATTACACCGT CAACAACAAG 1841AGAAATGGAG TGGCCAAAGT CAACATCTTC TTCAAGGAGC 1881TGAACTACAA AACCAATTCT GAGTCTCCCT CTGTCACGAT 1921GGTCACCCTC CTGTCCAACC TGGGCAGCCA GTGGAGCCTG 1961TGGTTCGGCT CCTCGGTGTT GTCTGTGGTG GAGATGGCTG 2001AGCTCGTCTT TGACCTGCTG GTCATCATGT TCCTCATGCT 2041GCTCCGAAGG TTCCGAAGCC GATACTGGTC TCCAGGCCGA 2081GGGGGCAGGG GTGCTCAGGA GGTAGCCTCC ACCCTGGCAT 2121CCTCCCCTCC TTCCCACTTC TGCCCCCACC CCATGTCTCT 2161GTCCTTGTCC CAGCCAGGCC CTGCTCCCTC TCCAGCCTTG 2201ACAGCCCCTC CCCCTGCCTA TGCCACCCTG GGCCCCCGCC 2241CATCTCCAGG GGGCTCTGCA GGGGCCAGTT CCTCC A CCTG 2281TCCTCTGGGG GGGCCCTGAG AGGGAAGGAG AGGTTTCTCA 2321CACCAAGGCA GATGCTCCTC TGGTGGGAGG GTGCTGGCCC 2361TGGCAAGATT GAAGGATGTG CAGGGCTTCC TCTCAGAGCC 2401GCCCAAACTG CCGTTGATGT GTGGAGGGGA AGCAAGATGG 2441GTAAGGGCTC AGGAAGTTGC TCCAAGAACA GTAGCTGATG 2481AAGCTGCCCA GAAGTGCCTT GGCTCCAGCC CTGTACCCCT 2521TGGTACTGCC TCTGAACACT CTGGTTTCCC CACCCAACTG 2561CGGCTAAGTC TCTTTTTCCC TTGGATCAGC CAAGCGAAAC 2601TTGGAGCTTT GACAAGGAAC TTTCCTAAGA AACCGCTGAT 2641AACCAGGACA AAACACAACC AAGGGTACAC GCAGGCATGC 2681ACGGGTTTCC TGCCCAGCGA CGGCTTAAGC CAGCCCCCGA 2721CTGGCCTGGC CACACTGCTC TCCAGTAGCA CAGATGTCTG 2761CTCCTCCTCT TGAACTTGGG TGGGAAACCC CACCCAAAAG 2801CCCCCTTTGT TACTTAGGCA ATTCCCCTTC CCTGACTCCC 2841GAGGGCTAGG GCTAGAGCAG ACCCGGGTAA GTAAAGGCAG 2881ACCCAGGGCT CCTCTAGCCT CATACCCGTG CCCTCACAGA 2921GCCATGCCCC GGCACCTCTG CCCTGTGTCT TTCATACCTC 2961TACATGTCTG CTTGAGATAT TTCCTCAGCC TGAAAGTTTC 3001CCCAACCATC TGCCAGAGAA CTCCTATGCA TCCCTTAGAA 3041CCCTGCTCAG ACACCATTAC TTTTGTGAAC GCTTCTGCCA 3081CATCTTGTCT TCCCCAAAAT TGATCACTCC GCCTTCTCCT 3121GGGCTCCCGT AGCACACTAT AACATCTGCT GGAGTGTTGC 3161TGTTGCACCA TACTTTCTTG TACATTTGTG TCTCCCTTCC 3201CAACTAGACT GTAAGTGCCT TGCGGTCAGG GACTGAATCT 3241TGCCCGTTTA TGTATGCTCC ATGTCTAGCC CATCATCCTG 3281CTTGGAGCAA GTAGGCAGGA GCTCAATAAA TGTTTGTTGC 3321ATGAAGGAAA AAAAAAAAAA AAAAA

The human epithelial Na+channel encoded by the SCNN1A cDNA with SEQ IDNO:24 has the following sequence (SEQ ID NO:25).

  1 MEGNKLEEQD SSPPQSTPGL MKGNKREEQG LGPEPAAPQQ  41PTAEEEALIE FHRSYRELFE FFCNNTTIHG AIRLVCSQHN  81RMKTAFWAVL WLCTFGMMYW QFGLLFGEYF SYPVSLNINL 121NSDKLVFPAV TICTLNPYRY PEIKEELEEL DRITEQTLFD 161LYKYSSFTTL VAGSRSRRDL RGTLPHPLQR LRVPPPPHGA 201RRARSVASSL RDNNPQVDWK DWKIGFQLCN QNKSDCFYQT 241YSSGVDAVRE WYRFHYINIL SRLPETLPSL EEDTLGNFIF 281ACRFNQVSCN QANYSHFHHP MYGNCYTFND KNNSNLWMSS 321MPGINNGLSL MLRAEQNDFI PLLSTVTGAR VMVHGQDEPA 361FMDDGGFNLR PGVETSISMR KETLDRLGGD YGDCTKNGSD 401VPVENLYPSK YTQQVCIHSC FQESMIKECG CAYIFYPRPQ 441NVEYCDYRKH SSWGYCYYKL QVDFSSDHLG CFTKCRKPCS 481VTSYQLSAGY SRWPSVTSQE WVFQMLSRQN NYTVNNKRNG 521VAKVNIFFKE LNYKTNSESP SVTMVTLLSN LGSQWSLWFG 561SSVLSVVEMA ELVFDLLVIM FLMLLRRFRS RYWSPGRGGR 601GAQEVASTLA SSPPSHFCPH PMSLSLSQPG PAPSPALTAP 641 PPAYATLGPR PSPGGSAGAS SST CPLGGP

Note that the underlined threonine at position 722 of the SEQ ID NO:23SCNN1A protein, and the underlined threonine at position 663 of the SEQID NO:25 SCNN1A protein, is threonine because some individuals havenucleotide sequence SEQ ID NO:22, where the variable nucleotide isadenine. However, position 722 of SEQ ID NO:23 and position 663 of SEQID NO:25 can be alanine in some individuals because those individualshave guanine as the variable nucleotide in sequence SEQ ID NO:22.

Patients with the threonine substitution in SCNN1A (adenine inrs2228576) have more functional Na⁺ channels and consequently higheractivity higher voltage currents across the cells. Hence, patients withsuch a threonine at the variable site in SCNN1A are more susceptible tohypertension than SCNN1A proteins with alanine at that position.Patients with the threonine substitution in SCNN1A can benefit fromadministration of amiloride.

Common and functional genetic variation of alpha adducin at amino acid460 has also been identified where some individuals have glycine andothers have tryptophan. A cDNA sequence for the human alpha adducin gene(ADD1) is available from the NCBI database as accession numberNM_001119.4 (GI:346644753). This ADD1 sequence is provided below as SEQID NO:26.

   1 GCACCCAGGT CGGGCGGTGG GGGCGAGCGG AGGGGCTGAG   41GGGCGGAGAG GCCTGGCGGG CCGCTGCTGC GGGCCAGGGG   81ACGGGGGCGG AGCCGGAGCC GGAGCCGACG GGCGGTGGCC  121GCACTGGGAC CCCGGAATCC CGCGCGCTGC CCACGATTCG  161CTTCTGAGGA ACCTAGAAAG ATTGTACAAT GAATGGTGAT  201TCTCGTGCTG CGGTGGTGAC CTCACCACCC CCGACCACAG  241CCCCTCACAA GGAGAGGTAC TTCGACCGAG TAGATGAGAA  281CAACCCAGAG TACTTGAGGG AGAGGAACAT GGCACCAGAC  321CTTCGCCAGG ACTTCAACAT GATGGAGCAA AAGAAGAGGG  361TGTCCATGAT TCTGCAAAGC CCTGCTTTCT GTGAAGAATT  401GGAATCAATG ATACAGGAGC AATTTAAGAA GGGGAAGAAC  441CCCACAGGCC TATTGGCATT ACAGCAGATT GCAGATTTTA  481TGACCACGAA TGTACCAAAT GTCTACCCAG CAGCTCCGCA  521AGGAGGGATG GCTGCCTTAA ACATGAGTCT TGGTATGGTG  561ACTCCTGTGA ACGATCTTAG AGGATCTGAT TCTATTGCGT  601ATGACAAAGG AGAGAAGTTA TTACGGTGTA AATTGGCAGC  641GTTTTATAGA CTAGCAGATC TCTTTGGGTG GTCTCAGCTT  681ATCTACAATC ATATCACAAC CAGAGTGAAC TCCGAGCAGG  721AACACTTCCT CATTGTCCCT TTTGGGCTTC TTTACAGTGA  761AGTGACTGCA TCCAGTTTGG TTAAGATCAA TCTACAAGGA  801GATATAGTAG ATCGTGGAAG CACTAATCTG GGAGTGAATC  841AGGCCGGCTT CACCTTACAC TCTGCAATTT ATGCTGCACG  881CCCGGACGTG AAGTGCGTCG TGCACATTCA CACCCCAGCA  921GGGGCTGCGG TCTCTGCAAT GAAATGTGGC CTCTTGCCAA  961TCTCCCCGGA GGCGCTTTCC CTTGGAGAAG TGGCTTATCA 1001TGACTACCAT GGCATTCTGG TTGATGAAGA GGAAAAAGTT 1041TTGATTCAGA AAAATCTGGG GCCTAAAAGC AAGGTTCTTA 1081TTCTCCGGAA CCATGGGCTC GTGTCAGTTG GAGAGAGCGT 1121TGAGGAGGCC TTCTATTACA TCCATAACCT TGTGGTTGCC 1161TGTGAGATCC AGGTTCGAAC TCTGGCCAGT GCAGGAGGAC 1201CAGACAACTT AGTCCTGCTG AATCCTGAGA AGTACAAAGC 1241CAAGTCCCGT TCCCCAGGGT CTCCGGTAGG GGAAGGCACT 1281GGATCGCCTC CCAAGTGGCA GATTGGTGAG CAGGAATTTG 1321AAGCCCTCAT GCGGATGCTC GATAATCTGG GCTACAGAAC 1361TGGCTACCCT TATCGATACC CTGCTCTGAG AGAGAAGTCT 1401AAAAAATACA GCGATGTGGA GGTTCCTGCT AGTGTCACAG 1441GTTACTCCTT TGCTAGTGAC GGTGATTCGG GCACTTGCTC 1481CCCACTCAGA CACAGTTTTC AGAAGCAGCA GCGGGAGAAG 1521ACAAGATGGC TGAACTCTGG CCGGGGCGAC GAAGCTTCCG 1561 AGGAA GGGCA GAATGGAAGC AGTCCCAAGT CGAAGACTAA 1601GTGGACTAAA GAGGATGGAC ATAGAACTTC CACCTCTGCT 1641GTCCCTAACC TGTTTGTTCC ATTGAACACT AACCCAAAAG 1681AGGTCCAGGA GATGAGGAAC AAGATCCGAG AGCAGAATTT 1721ACAGGACATT AAGACGGCTG GCCCTCAGTC CCAGGTTTTG 1761TGTGGTGTAG TGATGGACAG GAGCCTCGTC CAGGGAGAGC 1801TGGTGACGGC CTCCAAGGCC ATCATTGAAA AGGAGTACCA 1841GCCCCACGTC ATTGTGAGCA CCACGGGCCC CAACCCCTTC 1881ACCACACTCA CAGACCGTGA GCTGGAGGAG TACCGCAGGG 1921AGGTGGAGAG GAAGCAGAAG GGCTCTGAAG AGAATCTGGA 1961CGAGGCTAGA GAACAGAAAG AAAAGAGTCC TCCAGACCAG 2001CCTGCGGTCC CCCACCCGCC TCCCAGCACT CCCATCAAGC 2041TGGAGGAAGA CCTTGTGCCG GAGCCGACTA CTGGAGATGA 2081CAGTGATGCT GCCACCTTTA AGCCAACTCT CCCCGATCTG 2121TCCCCTGATG AACCTTCAGA AGCACTCGGC TTCCCAATGT 2161TAGAGAAGGA GGAGGAAGCC CATAGACCCC CAAGCCCCAC 2201TGAGGCCCCT ACTGAGGCCA GCCCCGAGCC AGCCCCAGAC 2241CCAGCCCCGG TGGCTGAAGA GGCTGCCCCC TCAGCTGTCG 2281AGGAGGGGGC CGCCGCGGAC CCTGGCAGCG ATGGGTCTCC 2321AGGCAAGTCC CCGTCCAAAA AGAAGAAGAA GTTCCGTACC 2361CCGTCCTTTC TGAAGAAGAG CAAGAAGAAG AGTGACTCCT 2401GAAAGCCCTG CGCTAACACT GTCCTGTCCG GAGCGACCCT 2441GGCTCTGCCA GCGTCCCCGG CCACGTCTGT GCTCTGTCCT 2481TGTGTAATGG AATGCAAAAA AGCCAAGCCC TCCGCCTAGA 2521GGTCCCCTCA CGTGACCAGC CCCGTGTAGC CCCGGGCTGA 2561CCCAGTGTGT GCTCAGCAGC CCCACCCCAC CCTGCCCCTT 2601GTCCTCTCAG AGCCTCAGCT TCTGGGGGAG ACATGCTCTC 2641CCCACAGGGG GGAGGCACTA AGTCATGGTC CTGGCTGGAA 2681GGTACTGAAG GCTTCTGCAG CTTTGGCTGC ACGTCACCCT 2721CCTGAGCCTC ACCTTTCCTG CCGTCCCTCC TGTTGTGAAA 2761TCACCACATT CTGTCTCTGC TTGGCTTCCC CTCCACCCTA 2801AAGTCTCAGG TGACGGACTC AGACTCCTGG CTTCATGTGG 2841CATTCTCTCT GCTCAGTGAT CTCACTTAAA TCTATATACA 2881AAGCCTTGGT CCCGTGAAAA CACTCGTGTG CCCACCAGCG 2921GCCTTGAAGA GGCAGGTCTG GGCCAGATGC TGGGCAGGAA 2961ACCCCAGCGG CAGATGGGCC TGTGTGCACC CAACGTGATG 3001CTATGCATGT CTGACCGACG ATCCCTCGAC CAGAATCAGA 3041TTCAGGAGCT CAGTTTCTTT TTCACTTGGG TCTCTGGATT 3081CCTGTCATAG GGAAGGTATA TCAGGAGGGG AAGAGGCCTT 3121TCTAGAATTT TCTTTGAGCA GGTTTACAAT TTAGCTTACA 3161TTTTTCGACT GTGAACGTGA ATAGGCTGCT TTTTGCTTTC 3201TTCTTTCCAG ACCCCACAGT AGAGCACTTT TCACTTATTT 3241GGGGGAGGCT TCAGGGGACT GTTCTCACCT TAACTCAGCC 3281AGAAAGATGC CCTAGTTGTG ATCAAAGGTA ACTCGAGGTG 3321GAGGGTAGCC CTGGGGCCCC TCGACATCAC CGTCATTGAT 3361GGAGCCTGAA CCGTGTGCTC CTCGGCAGAT GCTGTTGTTG 3401TTACTTCCCT CCAAGAGGCT GGAAAAGGGC TCAGAGCTGC 3441TGAGCAGGAA CCGGAGGGTG ACCCATTTCA GGAGGTGCCG 3481GTACCAGCCT GACTAGGTAC AGGCAAGCTT GTGTGGGCCC 3521AACAGGCCCT TGGTAGAGCT GGTGCCAGAT GTGGGCTCAG 3561ATCCTGGGCA TGATGGGCCG AGCCACCTCG GATCCCACTG 3601ATTGGCCAGC CGAGCGAGAA CCAGGCTGCT GCATGGCACT 3641GACCGCCGCT TCCAGCTTCC TCTGAGCCGC AGGGCCTGCT 3681ACGCGGGCAA GCGTGCTGCC TCTCTTCTGT GTCGTTTTGT 3721TGCCAAGGCA GAATGAAAAG TCCTTAACCG TGGACTCTTC 3761CTTTATCCCC TCCTTTACCC CACATATGCA ATGACTTTTA 3801ATTTTCACTT TTGTAGTTTA ATCCTTTGTA TTACAACATG 3841AAATATAGTT GCATATATGG ACACCGACTT GGGAGGACAG 3881GTCCTGAATG TCCTTTCTCC AGTGTAACAT GTTTTACTCA 3921CAAATAAAAT TCTTTCAGCA AGTTCCTTGT CTAAAAAAAA 3961 AAAAAAAAAA

The rs4961 single nucleotide polymorphism (SNP) is present in the ADD1gene, where the variable nucleotide is at about position 1566 in SEQ IDNO:26 (underlined), which can be guanine in some individuals and thyminein others. The rs4961 sequence (SEQ ID NO:27) is shown below, where theunderlined G/T is the SNP.

CCGGGGCGACGAAGCTTCCGAGGAA[G/T]GGCAGAATGGAAGCAGTCCC AAGTC.

The human alpha adducin encoded by the ADD1 cDNA with SEQ ID NO:26 hasthe following sequence (SEQ ID NO:28).

  1 MNGDSRAAVV TSPPPTTAPH KERYFDRVDE NNPEYLRERN  41MAPDLRQDFN MMEQKKRVSM ILQSPAFCEE LESMIQEQFK  81KGKNPTGLLA LQQIADFMTT NVPNVYPAAP QGGMAALNMS 121LGMVTPVNDL RGSDSIAYDK GEKLLRCKLA AFYRLADLFG 161WSQLIYNHIT TRVNSEQEHF LIVPFGLLYS EVTASSLVKI 201NLQGDIVDRG STNLGVNQAG FTLHSAIYAA RPDVKCVVHI 241HTPAGAAVSA MKCGLLPISP EALSLGEVAY HDYHGILVDE 281EEKVLIQKNL GPKSKVLILR NHGLVSVGES VEEAFYYIHN 321LVVACEIQVR TLASAGGPDN LVLLNPEKYK AKSRSPGSPV 361GEGTGSPPKW QIGEQEFEAL MRMLDNLGYR TGYPYRYPAL 401REKSKKYSDV EVPASVTGYS FASDGDSGTC SPLRHSFQKQ 441 QREKTRWLNS GRGDEASEE G QNGSSPKSKT KWTKEDGHRT 481 STSAVPNLFV PLNTNPKEVQ EMRNKIREQN LQDIKTAGPQ521 SQVLCGVVMD RSLVQGELVT ASKAIIEKEY QPHVIVSTTG 561PNPFTTLTDR ELEEYRREVE RKQKGSEENL DEAREQKEKS 601PPDQPAVPHP PPSTPIKLEE DLVPEPTTGD DSDAATFKPT 641LPDLSPDEPS EALGFPMLEK EEEAHRPPSP TEAPTEASPE 681PAPDPAPVAE EAAPSAVEEG AAADPGSDGS PGKSPSKKKK 721 KFRTPSFLKK SKKKSDS

Note that the underlined glycine at position 460 of the SEQ ID NO:28alpha adducin protein is glycine because some individuals havenucleotide sequence SEQ ID NO:26, where the variable nucleotide atposition 1566 is guanine. However, position 460 of SEQ ID NO:28 can betryptophan in some individuals because those individuals have thymine asthe variable nucleotide at position 1566 in sequence SEQ ID NO:28.

Individuals with the tryptophan variant of alpha adducin are more likelyto be salt sensitive, more likely to have hypertension and have agreater response to diuretics.

Genetic variation of the sodium (Na⁺) chloride (Cl⁻) co-transporter(SLC12A3) also has blood pressure consequences. A cDNA sequence for thesodium (Na⁺) chloride (Cl⁻) co-transporter (SLC12A3) is available fromthe NCBI database as accession number NM_000339.2 (GI:186910314). ThisSLC12A3 cDNA sequence is provided below as SEQ ID NO:29.

   1 CTGGCCCCTC CCTGGACACC CAGGCGACAA TGGCAGAACT   41GCCCACAACA GAGACGCCTG GGGACGCCAC TTTGTGCAGC   81GGGCGCTTCA CCATCAGCAC ACTGCTGAGC AGTGATGAGC  121CCTCTCCACC AGCTGCCTAT GACAGCAGCC ACCCCAGCCA  161CCTGACCCAC AGCAGCACCT TCTGCATGCG CACCTTTGGC  201TACAACACGA TCGATGTGGT GCCCACATAT GAGCACTATG  241CCAACAGCAC CCAGCCTGGT GAGCCCCGGA AGGTCCGGCC  281CACACTGGCT GACCTGCACT CCTTCCTCAA GCAGGAAGGC  321AGACACCTGC ATGCCCTGGC CTTTGACAGC CGGCCCAGCC  361ACGAGATGAC TGATGGGCTG GTGGAGGGCG AGGCAGGCAC  401CAGCAGCGAG AAGAACCCCG AGGAGCCAGT GCGCTTCGGC  441TGGGTCAAGG GGGTGATGAT TCGTTGCATG CTCAACATTT  481GGGGCGTGAT CCTCTACCTG CGGCTGCCCT GGATTACGGC  521CCAGGCAGGC ATCGTCCTGA CCTGGATCAT CATCCTGCTG  561TCGGTCACGG TGACCTCCAT CACAGGCCTC TCCATCTCAG  601CCATCTCCAC CAATGGCAAG GTCAAGTCAG GTGGCACCTA  641CTTCCTCATC TCCCGGAGTC TGGGCCCAGA GCTTGGGGGC  681TCCATCGGCC TCATTTTCGC TTTCGCCAAT GCCGTGGGTG  721TGGCCATGCA CACGGTGGGC TTTGCAGAGA CCGTGCGGGA  761CCTGCTCCAG GAGTATGGGG CACCCATCGT GGACCCCATT  801 AACGACATCC GCATCATTG G CGTGGTCTCG GTCACTGTGC  841 TGCTGGCCAT CTCCCTGGCT GGCATGGAGT GGGAGTCCAA 881 GGCCCAGGTG CTGTTCTTCC TTGTCATCAT GGTCTCCTTT  921GCCAACTATT TAGTGGGGAC GCTGATCCCC CCATCTGAGG  961ACAAGGCCTC CAAAGGCTTC TTCAGCTACC GGGCGGACAT 1001TTTTGTCCAG AACTTGGTGC CTGACTGGCG GGGTCCAGAT 1041GGCACCTTCT TCGGAATGTT CTCCATCTTC TTCCCCTCGG 1081CCACAGGCAT CCTGGCAGGG GCCAACATAT CTGGTGACCT 1121CAAGGACCCT GCTATAGCCA TCCCCAAGGG GACCCTCATG 1161GCCATTTTCT GGACGACCAT TTCCTACCTG GCCATCTCAG 1201CCACCATTGG CTCCTGCGTG GTGCGTGATG CCTCTGGGGT 1241CCTGAATGAC ACAGTGACCC CTGGCTGGGG TGCCTGCGAG 1281GGGCTGGCCT GCAGCTATGG CTGGAACTTC ACCGAGTGCA 1321CCCAGCAGCA CAGCTGCCAC TACGGCCTCA TCAACTATTA 1361CCAGACCATG AGCATGGTGT CAGGCTTCGC GCCCCTGATC 1401ACGGCTGGCA TCTTCGGGGC CACCCTCTCC TCTGCCCTGG 1441CCTGCCTTGT CTCTGCTGCC AAAGTCTTCC AGTGCCTTTG 1481CGAGGACCAG CTGTACCCAC TGATCGGCTT CTTCGGCAAA 1521GGCTATGGCA AGAACAAGGA GCCCGTGCGT GGCTACCTGC 1561TGGCCTACGC CATCGCTGTG GCCTTCATCA TCATCGCTGA 1601GCTCAACACC ATAGCCCCCA TCATTTCCAA CTTCTTCCTC 1641TGCTCCTATG CCCTCATCAA CTTCAGCTGC TTCCACGCCT 1681CCATCACCAA CTCGCCTGGG TGGAGACCTT CATTCCAATA 1721CTACAACAAG TGGGCGGCGC TGTTTGGGGC TATCATCTCC 1761GTGGTCATCA TGTTCCTCCT CACCTGGTGG GCGGCCCTCA 1801TCGCCATTGG CGTGGTGCTC TTCCTCCTGC TCTATGTCAT 1841CTACAAGAAG CCAGAGGTAA ATTGGGGCTC CTCGGTACAG 1881GCTGGCTCCT ACAACCTGGC CCTCAGCTAC TCGGTGGGCC 1921TCAATGAGGT GGAAGACCAC ATCAAGAACT ACCGCCCCCA 1961GTGCCTGGTG CTCACGGGGC CCCCCAACTT CCGCCCGGCC 2001CTGGTGGACT TTGTGGGCAC CTTCACCCGG AACCTCAGCC 2041TGATGATCTG TGGCCACGTG CTCATCGGAC CCCACAAGCA 2081GAGGATGCCT GAGCTCCAGC TCATCGCCAA CGGGCACACC 2121AAGTGGCTGA ACAAGAGGAA GATCAAGGCC TTCTACTCGG 2161ATGTCATTGC CGAGGACCTC CGCAGAGGCG TCCAGATCCT 2201CATGCAGGCC GCAGGTCTCG GGAGAATGAA GCCCAACATT 2241CTGGTGGTTG GGTTCAAGAA GAACTGGCAG TCGGCTCACC 2281CGGCCACAGT GGAAGACTAC ATTGGCATCC TCCATGATGC 2321CTTTGATTTC AACTATGGCG TGTGTGTCAT GAGGATGCGG 2361GAGGGACTCA ACGTGTCCAA GATGATGCAG GCGCACATTA 2401ACCCCGTGTT TGACCCAGCG GAGGACGGGA AGGAAGCCAG 2441CGCCAGAGGT GCCAGGCCAT CAGTCTCTGG CGCTTTGGAC 2481CCCAAGGCCC TGGTGAAGGA GGAGCAGGCC ACCACCATCT 2521TCCAGTCGGA GCAGGGCAAG AAGACCATAG ACATCTACTG 2561GCTCTTTGAC GATGGAGGCC TCACCCTCCT CATTCCCTAT 2601CTCCTTGGCC GCAAGAGGAG GTGGAGCAAA TGCAAGATCC 2641GTGTGTTCGT AGGCGGCCAG ATTAACAGGA TGGACCAGGA 2681GAGAAAGGCG ATCATTTCTC TGCTGAGCAA GTTCCGACTG 2721GGATTCCATG AAGTCCACAT CCTCCCTGAC ATCAACCAGA 2761ACCCTCGGGC TGAGCACACC AAGAGGTTTG AGGACATGAT 2801TGCACCCTTC CGTCTGAATG ATGGCTTCAA GGATGAGGCC 2841ACTGTCAACG AGATGCGGCG GGACTGCCCC TGGAAGATCT 2881CAGATGAGGA GATTACGAAG AACAGAGTCA AGTCCCTTCG 2921GCAGGTGAGG CTGAATGAGA TTGTGCTGGA TTACTCCCGA 2961GACGCTGCTC TCATCGTCAT CACTTTGCCC ATAGGGAGGA 3001AGGGGAAGTG CCCCAGCTCG CTGTACATGG CCTGGCTGGA 3041GACCCTGTCC CAGGACCTCA GACCTCCAGT CATCCTGATC 3081CGAGGAAACC AGGAAAACGT GCTCACCTTT TACTGCCAGT 3121AACTCCAGGC TTTGACATCC CTGTCCACAG CTCTGAGTGT 3161GTGGGATAAG TTGGAACTTG ATTGCCTCTA GTCCACAGGG 3201ATGAGACTCA TGTTCTGTTG CACTTTAAGT GGCAGCATCT 3241GATGATCTCA CCGAAAAAGA TGGTAGATTT CCAAATCTGG 3281CTGGACTCCA CTTCCATGGG ACACATTCCC TGGGTCTTGT 3321GTTTATAGGC TAGAGAAATA GCAGATGGAG CTGCAAGGAA 3361AACTCTCTAA AGCATCCTAT TCCTTTTAAA GGATTTCTTT 3401TGATTTTGAT GACCATTAAT TAAGAGTTCA GTCTTTGATT 3441TGTATGCAAA TTGGAGTCCC AATGCTGGGC GTGAATCTTG 3481ACAGTTTCTA CAGACCTTCC TGGGTGAAAG TTCCTAAATC 3521ATGCCCTGCT TCCTCCAATA GGAGAATGGG AGCCTCACCT 3561GTAGGACCTA CAGGCTCTCT AAGGAATGCA GGTCTCTCTC 3601TGAGCCTCCA CAGCCAGGCA AATACATATA TATATATTTT 3641TTTTTTAGAT GAAGTTTTTT CTCTTGTTGC CCAGGCTAGG 3681GTGTAATGGC ATGATCTCAG GTCACTGCAA CCTCCTCCCG 3721GGTTCAAGCA TTTCTTCTGT CTCAGCCTCC CGAATAGCTG 3761GGATTACAGG CACCTGCCAT CACACGAGCT AATTTTTGTA 3801TTTTTAGTAG AGATGGGGTT TCACCATGTT GACCAGGCTG 3841GTGTTGAGCT CCTGACCTCA GGTGATCCAC CCACCTCGGT 3881CTCCCAAAGT GCTGGGGTTA CAGGCCTGAG CCACTGCGCC 3921CGGCCCAGGC AAATTTCTTG AACCACTTCT CACTCCCGTC 3961ACTTTCAATA AGGGGTCTTT GATGTCTTCA CTGGTTCTTT 4001GGACGAGGGA CTTTTCGAAC TTTTTTGGTT GCAACACACA 4041GTAAGAAATA TACTTCACAC TGAGACTTGC AGCGCACACA 4081CACGGAAACG ACCAAAACAA AAATGTCACA AAACAATACT 4121TACCCTTCCC TGGGGGACGT CCTCCAGTAT GTTCTGTTCT 4161GTTTATTTTT CACTGTTGGT TGCAATCCAA TAAAATGACT 4201TTGGGATCCA CTCATGGGTG GGGACCCACA CATTTGAAAG 4241GCATGGCCAC CTTTCTGTTG TGCCTTGCAT TTGTCCACAC 4281ACAGGGAGTC TGGCTGAGCT GGGGAAAGGC CACGGCTGGG 4321TGTCATTGCC ATTTTCCCAG CTCATCTCAC CGGGAAGAAA 4361AGCAGATTGA CAGAACACGT GAGGAGGGGT ATTGATGGCA 4001GGAGAGTCAA AAAAGAGTTT TAAAGAAGGG GCAAGGTTGA 4441AGGAGTCTAG TGGCAAGGGT AAGATTTCAG GCATGGTTAA 4481GAACAGACGA CAAGGATGTC AGGAATGAAG ATGTGGAGAG 4521GGGTGTAGAG ATGGCAAGGT TGGCAAGGAA CAGATAGGCA 4561GGAGCAGGTC CAAGCCAAGC CTAGCCCAAG ACCAGGTGAA 4601AGGAGAGGGG AGGAGGAGCC ACCTGCAAGA GATGGAAAGA 4641GCAGGCGGCA GAGGGGGCTG GCAGGGAGGG GCTGTTAAGA 4681GTGGGGTTGG AGGTGGGAGA GAAGCTAGGA CAAGGGAGAT 4721GGAGAAAGGA CCTATACCTG GCTCACGGAA GGCCTTCAGG 4761TCACTACACG TTGAACATCC CCAGTGTTTG AGCCCCCAAA 4801GCTAGGGTGC AAGAGCACTG CCATCGAATG CCAGTGGGTG 4841AGGCCAAGTG AGGGTATTTG CAGCTCTAGA CATAACCAAG 4881AAGCGTAAAG GTGAGTTGTT TGGTGGTACG ACTGCCTGTG 4921CCTTCTTCCG ATGGCACTGG GGTGGCTGAA GGAACAGACA 4961TCTTTGGGTT TCATCAGCCT CCTCCAAGAC TGCTGCAGTG 5001CCTACACTTT AGACTTCAGA AGGAGACTAA AGACTTCTAG 5041AATTTAGAAG GAGATCTGAA GTCTCCTTTC TGGAGTTACA 5081ACCCAAAGGA TGTTAGCATT TCTCAGGTCA TCCCACTGCA 5121AAGCCCAGAA GGCTTGGGGC TCCCAGGCTG CTCTGAAGCC 5161CCACTGTCTG ACCGCCTCAG GGCTTGCTAC GAGGGACTGG 5201GGCACGGCCA AGCTGACTAG GAACAGCTCT CGTGCTCCTG 5241AGGGACCTGG AGGATGGGCC TGCCTCCCAG CCATTGAGCT 5281GGATTCTGGG ATAATTCTTA ACTCGAAATA AGGGGAAGCA 5321TCCATCAGGG AATGCTGGCC TTTCTAGAGC CACGTAGAAA 5361ACAATTTTCT GGTTCTTCAA ACCTCAAAGA GTCCTTGGTC 5401CAAAAAACAG AATGTTTTGG CTTCGGGTGT CAAAAAAAAA 5441ATTTTCACGA TGTCAGAAAT AGTATGTTTT TAACAATAGT 5481AATAGCTTTG TAAAAAAATA AAAAGCTTTA ACAGCGAGGC 5521CATAAACAAT GAAATGAATA AAAACGGTGG TCATTCAGTC 5561AACGGAAAAA AAAAAAAAAA AA

The rs1529927 single nucleotide polymorphism (SNP) is present in theSLC12A3 gene, where the variable nucleotide is at about position 820 inSEQ ID NO:29 (underlined), which can be guanine in some individuals andcytosine in others. The rs1529927 sequence (SEQ ID NO:30) is shownbelow, where the underlined C/G is the SNP.

CCCATTAACGACATCCGCATCATTG[C/G]CGtGGTCTCGGTCACTGTG CTGCTG.

The human the sodium (Na⁺) chloride (Cl⁻) co-transporter encoded by theSLC12A3 cDNA with SEQ ID NO:29 has the following sequence (SEQ IDNO:31).

   1 MAELPTTETP GDATLCSGRF TISTLLSSDE PSPPAAYDSS   41HPSHLTHSST FCMRTFGYNT IDVVPTYEHY ANSTQPGEPR   81KVRPTLADLH SFLKQEGRHL HALAFDSRPS HEMTDGLVEG  121EAGTSSEKNP EEPVRFGWVK GVMIRCMLNI WGVILYLRLP  161WITAQAGIVL TWIIILLSVT VTSITGLSIS AISTNGKVKS  201GGTYFLISRS LGPELGGSIG LIFAFANAVG VAMHTVGFAE  241TVRDLLQEYG APIVDPINDI RII G VVSVTV LLAISLAGME  281WESKAQVLFF LVIMVSFANY LVGTLIPPSE DKASKGFFSY  321RADIFVQNLV PDWRGPDGTF FGMFSIFFPS ATGILAGANI  361SGDLKDPAIA IPKGTLMAIF WTTISYLAIS ATIGSCVVRD  401ASGVLNDTVT PGWGACEGLA CSYGWNFTEC TQQHSCHYGL  441INYYQTMSMV SGFAPLITAG IFGATLSSAL ACLVSAAKVF  481QCLCEDQLYP LIGFFGKGYG KNKEPVRGYL LAYAIAVAFI  521IIAELNTIAP IISNFFLCSY ALINFSCFHA SITNSPGWRP  561SFQYYNKWAA LFGAIISVVI MFLLTWWAAL IAIGVVLFLL  601LYVIYKKPEV NWGSSVQAGS YNLALSYSVG LNEVEDHIKN  641YRPQCLVLTG PPNFRPALVD FVGTFTRNLS LMICGHVLIG  681PHKQRMPELQ LIANGHTKWL NKRKIKAFYS DVIAEDLRRG  721VQILMQAAGL GRMKPNILVV GFKKNWQSAH PATVEDYIGI  761LHDAFDFNYG VCVMRMREGL NVSKMMQAHI NPVFDPAEDG  801KEASARGARP SVSGALDPKA LVKEEQATTI FQSEQGKKTI  841DIYWLFDDGG LTLLIPYLLG RKRRWSKCKI RVFVGGQINR  881MDQERKAIIS LLSKFRLGFH EVHILPDINQ NPRAEHTKRF  921EDMIAPFRLN DGFKDEATVN EMRRDCPWKI SDEEITKNRV  961KSLRQVRLNE IVLDYSRDAA LIVITLPIGR KGKCPSSLYM 1001AWLETLSQDL RPPVILIRGN QENVLTFYCQ

Note that the underlined glycine at position 264 of the SEQ ID NO:31sodium (Na⁺) chloride (Cl⁻) co-transporter protein is glycine becausesome individuals have nucleotide sequence SEQ ID NO:29, where thevariable nucleotide at position 820 is guanine. However, position 264 ofSEQ ID NO:29 can be alanine in some individuals because thoseindividuals have cytosine as the variable nucleotide at position 820 insequence SEQ ID NO:29.

Patients with the alanine variant of SLC12A3 (encoded by the rs1529927site (SEQ ID NO:30)) exhibit a stronger diuretic effect to loopdiuretics and demonstrate more excretion of Cl⁻ and K⁺ in response totherapy. Hence, subject with alanine or guanine at the rs1529927 siteare more response to diuretics.

The WNK1 gene has functional and common polymorphisms that affect how asubject's blood pressure responds to drugs. Several cDNA sequences forthe WNT1 gene are available from the NCBI database.

The rs2107614 single nucleotide polymorphism (SNP) is present in anintron of the WNK1 gene, where the variable nucleotide can be thymine insome individuals and cytosine in others. The rs2107614 sequence (SEQ IDNO:33) is shown below, where the underlined C/T is the SNP.

CACTTCCTCCAAAAAAAAAGAAAAC[C/T]CCATTTCCCCTCAACTCTT CCAGTT.

Another SNP, rs1159744, is present an intron of the WNK1 gene, where thevariable nucleotide can be guanine in some individuals and cytosine inothers. The rs1159744 sequence (SEQ ID NO:34) is shown below, where theunderlined C/G is the SNP.

AATGTTAACAGTATAGAAAATTTTA[C/G]CTCAACAAATAGAGAATAT CAGTAA.

Patients with the cytosine variant of WNK1 at SNP positions rs1159744and rs2107614 exhibit greater blood pressure reductions in response toloop diuretic therapy when compared to patients with the guanine orthymine variants at these two sites, respectively (Turner et al.,Hypertension 46:758-765 (2005)).

Therapy

The methods, reagents, devices, and kits described herein can be usedfor determining whether a subject may benefit from treatment with ablood pressure medication, and which medication can be more effectivefor treating high blood pressure. For example, the methods describedherein can be employed for determining whether a subject should betreated with a diuretic, an angiotensin converting enzyme (ACE)inhibitor, or a beta-blocker. Such determination is performed byidentifying or detecting whether the subject has a genetic variant orsingle nucleotide polymorphism in his or her ADRB1, ADRB2, CYP2D6,angiotensin converting enzyme (ACE), angiotensinogen, angiotensinreceptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺)chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1polypeptides or nucleic acids. If testing of the subject's tissue sampleshows that the subject has a genetic variant or single nucleotidepolymorphism in his or her ADRB1, ADRB2, CYP2D6, angiotensin convertingenzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻)co-transporters (such as SLC12A3), and/or WNK1 polypeptides or nucleicacids, a suitable therapeutic regimen can be prescribed for the subject.

A diuretic promotes the production or urine. Diuretics are sometimesgrouped into three categories: thiazides, loop, and potassium-sparingdiuretics. Thiazide diuretics include chlorothiazide,hydrochlorothiazide, indapamide, metolazone, and chlorthalidone. Loopdiuretics include furosemide, bumetanide, ethacrynic acid, andtorsemide. Examples of potassium-sparing diuretics include amiloride,eplerenone, spironolactone, and triamterene.

Examples of diuretics that can be employed also include furosemide,thiazides, carbonic anhydrase inhibitors, potassium-sparing diuretics(e.g., aldosterone antagonists, spironolactone, eplerenone, potassiumcanreonate, amiloride, triamterene, aldactone, and combinationsthereof), calcium-sparing diuretics. For example, the diuretic can beacetazolamide, amiloride, bumetanide, chlorothalidone, chlorothiazide,ethacrynic acid, furosemide, glycerin, hydrochlorothiazide,hydroflumthiazide, indapamide, isosorbide, mannitol, methazolamide,methylchlothiazide, metolazone, dichlorphenamide, spironolactone,torsemide, triamterene, urea, and combinations thereof.

The angiotensin converting enzyme inhibitor can be selected fromenalapril, lisinopril, captopril alacipril, benazapril, cilazapril,delapril, fosinopril, perindopril, quinapril, ramipril, moveltipril,spirapril, ceronapril, imidapril, temocapril, trandolopril, utilbapril,zofenopril, CV5975, EMD 56855, libenzapril, zalicipril, HOE065, MDL27088, AB47, DU 1777, MDL 27467A, Equaten™, Prentyl™, Synecor™, andY23785; and the diuretic is selected from hydrochlorothiazide (HCTZ),furosemide, altizide, trichlormethazide, triflumethazide, bemetizide,cyclothiazide, methylchlothiazide, azosemide, chlorothiazide, butizide,bendroflumethazide, cyclopenthiazide, benzclortriazide, polythiazide,hydroflumethazide, benzthiazide, ethiazide, penflutazide, and anycombination thereof.

The angiotensin II receptor antagonists can, for example, be losartan,valsartan, candesartan, irbesartan, olmesartan, or any combinationthereof.

The renin inhibitors can be urea derivatives of di- and tri-peptides(See U.S. Pat. No. 5,116,835), amino acids and derivatives (U.S. Pat.Nos. 5,095,119 and 5,104,869), amino acid chains linked by non-peptidicbonds (U.S. Pat. No. 5,114,937), di- and tri-peptide derivatives (U.S.Pat. No. 5,106,835), peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and4,845,079) and peptidyl beta-aminoacyl aminodiol carbamates (U.S. Pat.No. 5,089,471); also, a variety of other peptide analogs as disclosed inthe following U.S. Pat. Nos. 5,071,837; 5,064,965; 5,063,207; 5,036,054;5,036,053; 5,034,512 and 4,894,437, and small molecule renin inhibitors(including diol sulfonamides and sulfinyls (U.S. Pat. No. 5,098,924),N-morpholino derivatives (U.S. Pat. No. 5,055,466), N-heterocyclicalcohols (U.S. Pat. No. 4,885,292) and pyrolimidazolones (U.S. Pat. No.5,075,451); also, pepstatin derivatives (U.S. Pat. No. 4,980,283) andfluoro- and chloro-derivatives of statone-containing peptides (U.S. Pat.No. 5,066,643), enalkrein, RO 42-5892, A 65317, CP 80794, ES1005, ES8891, SQ 34017, aliskiren((2S,4S,5S,7S)—N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)phenyl]-octanamidhemifumarate) SPP600, SPP630 and SPP635), or any combination thereof.

Other therapeutic agents can also be administered including endothelinreceptors antagonists, vasodilators, calcium channel blockers (e.g.,amlodipine, nifedipine, veraparmil, diltiazem, gallopamil, nifedipine,nimodipins, nicardipine), potassium channel activators (e.g.,nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam),diuretics (e.g., hydrochlorothiazide), sympatholitics, beta-adrenergicblocking drugs (e.g., propranolol, atenolol, bisoprolol, carvedilol,metoprolol, or metoprolol tartrate), alpha adrenergic blocking drugs(e.g., doxazocin, prazocin or alpha methyldopa) central alpha adrenergicagonists, peripheral vasodilators (e.g. hydralazine), lipid loweringagents (e.g., simvastatin, lovastatin, ezetamibe, atorvastatin,pravastatin), metabolic altering agents including insulin sensitizingagents and related compounds (e.g., muraglitazar, glipizide, metformin,rosiglitazone) or with other drugs beneficial for the prevention or thetreatment of disease including nitroprusside and diazoxide.

The therapeutic protocol can generally be conducted as follows. An assayof all sixteen genotypes (polymorphic sites) can be performed. Thetherapeutic decision tree of the results can be as follows.

To ascertain whether a diuretic should be administered to a subject, thefollowing can be performed.

-   -   If the subject is (a) homozygous for cytosine at the rs1529927        (SEQ ID NO:30) variable site (expressing alanine at position 264        of the SLC12A3 gene product); (b) homozygous for adenine at the        rs2228576 (SEQ ID NO:22) variable site (expressing threonine at        about 663 or 722 of the SCNN1A protein); and/or (c) homozygous        for thymine at the rs4961 (SEQ ID NO:27) variable site        (expressing tryptophan at about position 460 of the adducin        protein) then the patient should initially start with a diuretic        as the first line of therapy. If the patient is heterozygous at        these sites, then genetic variation within the polymorphic sites        relating to vasodilator and beta-blocker drug class responses        should initially be considered.    -   If the patient does not carry homozygous variants that are known        to be functionally important within the vasodilator and        beta-blocker classes, but are heterozygous at rs1529927,        rs2228576, and rs4961, then diuretic therapy should initially be        considered as first-line therapy.    -   If the subject is homozygous for cytosine at the WNK1 rs1159744        (SEQ ID NO:34) variable site and also homozygous for cytosine at        the WNK1 rs2107614 (SEQ ID NO:33) variable site then the patient        should start with a loop diuretic as first-line of therapy.    -   If the patient does not carry homozygous variants within the        vasodilator and beta-blocker classes that are known to be        functionally important, but are heterozygous at rs1529927,        rs2228576, and rs4961, then loop diuretic therapy should        initially be considered as first-line therapeutic agent.

To ascertain whether a vasodilator should be administered to a subjectthe following can be performed.

-   -   If the subject is homozygous for cytosine at the rs5186 (SEQ ID        NO:16) variable site of AGT1R, and the subject is homozygous        cytosine at the rs12750834 (SEQ ID NO:20) variable site of        renin, then the patient should use an angiotensin II (AII)        receptor blocker as a first line of therapy.    -   If the patient is heterozygous for cytosine at the rs5186 and        rs12750834 variable sites, but does not present with other        important functional genotypes within the diuretic and        beta-blockade classes, then the patient should also use an        angiotensin II receptor blocker as a first line of therapy.    -   If the patient is homozygous for cytosine at the rs699 (SEQ ID        NO:14) variable site of AGT, or for the deletion at the        rs1799752 (SEQ ID NO:12; SEQ ID NO:35) of ACE, then the patient        will likely benefit most from an angiotensin-converting enzyme        (ACE) inhibitor.    -   Patients who are homozygous for cytosine at the rs699 (SEQ ID        NO:14) will likely benefit most from BOTH ACE inhibition and        angiotensin II (AII) receptor blockade.    -   Patients who are heterozygous for the deletion at the rs1799752        (SEQ ID NO:12; SEQ ID NO:35) and heterozygous for cytosine at        the rs699 (SEQ ID NO:14) variable site should be administered        other drug classes (e.g., diuretic initially followed by        beta-blockade). Although homozygosity at other sites has a        greater impact on hypertension than heterozygosity at rs1799752        and rs699, this is generally true only if the patient has        combined homozygosity at sites indicating that drug classes        other than vasodilators should be administered.

To ascertain whether a beta-blocker should be administered to a subjectthe following can be performed.

-   -   Patients homozygous for adenine at the rs3892097 (SEQ ID NO:10)        variable site of the CYP2D6 gene should initially consider the        use of atenolol and carevdilol as therapy. This is PARTICULARLY        important if the patient is homozygous for cytosine at the        rs1801253 (SEQ ID NO:3) variable site (and expresses arginine at        position 389 of the β₁AR polypeptide), or if the patient is        homozygous for adenine at the rs1801252 variable position (and        expresses serine at position 49 of the β₁AR polypeptide).    -   The rs1042713 (SEQ ID NO:6) and rs1042714 (SEQ ID NO:7) variable        sites are less important of the other polymorphism sites within        the beta-blocker class of drugs and generally indicate patients        who will likely respond to non-selective beta-blockade. Thus,        subjects who are homozygous for guanine at the rs1042713        variable site (and express glycine at about position 16 of the        ADRB2 gene product) as well as subjects who are homozygous for        guanine at the rs1042714 position (and express glutamic acid at        β₂AR position 27) are the most responsive to beta-blocker drugs.    -   If subjects are non-homozygous for polymorphisms in the        beta-blockade class of variants, but are homozygous for cytosine        at the rs1801253 (SEQ ID NO:3) variable site (and expresses        arginine at position 389 of the β₁AR polypeptide), or if        subjects are homozygous for adenine at the rs1801252 variable        position (and expresses serine at position 49 of the β₁AR        polypeptide), the beta-blockade class should be considered a        possible line of therapy if they do not carry functional        mutations within the diuretic and vasodilator classes of drugs.        Polymorphism Detection

The polymorphism present in genes such as ADRB1, ADRB2, cytochrome P4502D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen,angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin,sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/orWNK1 can be detected by any available procedure. For example, samples ofcDNA, genomic DNA, and/or mRNA can be obtained from a subject and thesequences of polymorphic or variant sites can be evaluated by proceduressuch as nucleic acid amplification (e.g., PCR), reverse transcription,insertion/deletion analysis, primer extension, probe hybridization, SNPanalysis, sequencing, restriction fragment length polymorphism,Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight massspectrometry (MALDI-TOF MS), Sequenom MassArray genotyping, Sangersequencing, polyacrylamide gel electrophoresis, agarose gelelectrophoresis, probe array hybridization analysis, and combinationsthereof.

The methods for detecting polymorphisms can therefore involve detectingan alteration in a nucleic acid. As used herein a “nucleic acid” is aDNA or RNA molecule. A nucleic acid can be a segment of genomic DNA(e.g., an entire gene, an intron of a gene, an exon of a gene, a segmentthat includes regulatory elements, a 5′ non-coding segment, a 3′non-coding segment, or any combination thereof). The nucleic acid canalso be a cDNA (having exons but not introns), an amplicon, an RNA, aprimer, or probe.

Probes and/or primers can be used that can hybridize to nucleic acidsegments flanking or including of any of SNPs, insertions, deletions,polymorphic, or other variant segments of ADRB1, ADRB2, cytochrome P4502D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen,angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin,sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/orWNK1 genes. For example, probes and/or primers can be employed thathybridize to nucleic acid segments flanking or including any of thefollowing polymorphisms: rs1801252 (ADRB1), rs1801253 (ADRB1), rs1042713(ADRB2), rs1042714 (ADRB2), rs3892097 (CYP2D6), rs1799752 (ACE), rs699(AGT), rs5186 (AGT1R), rs12750834 (renin), rs2228576 (SCNN1A), rs4961(ADD1), rs1529927 (SLC12A3), rs2107614 (WNK1), or rs1159744 (WNK1). Forexample, the probes and/or primers can separately hybridize to segmentsof any of SEQ ID NO:2, 3, 6, 7, 10, 12, 14, 16, 19, 20, 22, 27, 30, 32,33, 34, as well as to the complementary sequences, amplicons, cDNA,cRNA, and genomic sequences thereof. The probes and/or primers canhybridize to genomic, complementary, amplicon, or cDNA sequences thatflank up to 50 nucleotides of any of SEQ ID NO:2, 3, 6, 7, 10, 12, 14,16, 19, 20, 22, 27, 30, 33, or 34, on either or both of the 5′ and 3′sides of the polymorphism.

Methods and devices described herein can detect at least two of thesepolymorphisms, or at least three of these polymorphisms, or at least offour of these polymorphisms, or at least five of these polymorphisms, orat least of six of these polymorphisms, or at least seven of thesepolymorphisms, or at least of eight of these polymorphisms, or at leastnine of these polymorphisms, or at least often of these polymorphisms,or at least eleven of these polymorphisms, or at least of twelve ofthese polymorphisms, or at least thirteen of these polymorphisms, or atleast fourteen of these polymorphisms, or at least fifteen of thesepolymorphisms or all of these polymorphisms. In some embodiments, themethods and devices described herein detect no other polymorphisms,although such methods and devices can include steps and probes fordetecting 1-4 control target nucleic acids. For example, the methods,devices, and kits described herein can detect and evaluate about sixteenpolymorphisms.

The probes and primers can be of any convenient length selected by oneof skill in the art such as at least 12 nucleotides long, or at least 13nucleotides long, or at least 14 nucleotides long, or at least 15nucleotides long, or at least 16 nucleotides long, or at least 17nucleotides long, or at least 18 nucleotides long, or at least 19nucleotides long, or at least 20 nucleotides long. In some embodiments,the probes and primers can be less than 150 nucleotides in length, orless than 125 nucleotides in length, or less than 100 nucleotides inlength, or less than 75 nucleotides in length, or less than 65nucleotides in length, or less than 60 nucleotides in length, or lessthan 55 nucleotides in length, or less than 50 nucleotides in length, orless than 45 nucleotides in length, or less than 40 nucleotides inlength.

To detect hybridization, it may be advantageous to employ probes,primers and other nucleic acids in combination with an appropriatedetection means. Labels incorporated into primers, incorporated into theamplified product during amplification, or attached to probes that canhybridize to the target, or its amplified product, are useful in theidentification of nucleic acid molecules. A number of different labelsmay be used for this purpose including, but not limited to,fluorophores, chromophores, radiolabels, enzymatic tags, antibodies,chemiluminescence, electroluminescence, and affinity labels. One ofskill in the art will recognize that these and other labels can be usedwith success in this invention.

Examples of affinity labels include, but are not limited to thefollowing: an antibody, an antibody fragment, a receptor protein, ahormone, biotin, dinitrophenyl (DNP), or any polypeptide/proteinmolecule that binds to an affinity label. Examples of enzyme tagsinclude enzymes such as urease, alkaline phosphatase or peroxidase tomention a few. Colorimetric indicator substrates can be employed toprovide a detection means visible to the human eye orspectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples. Examples of fluorophoresinclude, but are not limited to, Alexa 350, Alexa 430, AMCA, BODIPY630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX,Cascade Blue, Cy2, Cy3, Cy5, 6-FAM, Fluorescein, HEX, 6-JOE, OregonGreen 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET, Tetramethylrhodamine,and Texas Red.

Means of detecting such labels are well known to those of skill in theart. For example, radiolabels may be detected using photographic film orscintillation counters. In other examples, fluorescent markers may bedetected using a photodetector to detect emitted light. In still furtherexamples, enzymatic labels are detected by providing the enzyme with asubstrate and detecting the reaction product produced by the action ofthe enzyme on the substrate, and colorimetric labels are detected bysimply visualizing the colored label or by use of spectrometer.

So called “direct labels” are detectable labels that are directlyattached to or incorporated into a probe or primer, or to the target(sample) nucleic acid prior to hybridization to a probe that can, forexample, be present on a plate, chip, microtiter plate, or microarray.In contrast, so called “indirect labels” are joined to the hybrid duplexafter hybridization. In some embodiments, the indirect label is attachedto a binding moiety that has been attached to the target nucleic acidprior to the hybridization. Thus, for example, the target nucleic acidmay be biotinylated before the hybridization. After hybridization, anavidin-conjugated fluorophore will bind the biotin-bearing hybridduplexes providing a label that is easily detected. For a detailedreview of methods of labeling nucleic acids and detecting labeledhybridized nucleic acids see, for example, Peter C. van der Vliet & ShivPillai, eds., Laboratory Techniques in Biochemistry and MolecularBiology (1993).

Probe arrays, assay plates, and gene chip technology provide a means ofrapidly screening a large number of nucleic acid samples for theirability to hybridize to a variety of probes immobilized on a solidsubstrate that is part of the probe array, assay plate, gene chip ormicroarray. The technology capitalizes on the complementary bindingproperties of single stranded nucleic acid probe to screen nucleic acidsamples by hybridization (Pease et al., Proc. Natl. Acad. Sci. U.S.A.91: 5022-5026 (1994); U.S. Patent to Fodor et al. (1991)). Basically, anucleic acid probe array or gene chip consists of a solid substrate withan attached array of single-stranded probe molecules. In someembodiments, the probes can fold back on (i.e., hybridize to) themselvesto quench a signal from an attached label, but the probes unfold tohybridize to a target nucleic acid, whereupon the signal from theattached label becomes detectable. In other embodiments, the probe canbe complementary to the segment of a target nucleic acid but the 3′ endof the probe terminates one nucleotide short of a SNP in the targetnucleic acid. The target nucleic acid can be longer than the probe. Asignal can be detected upon primer extension of the probe, where theassay mixture contains just one type of labeled nucleotide that can basepair with the variant nucleotide of the SNP. After washing, the presenceor absence of the SNP is detectable by incorporation ornon-incorporation of the labeled SNP nucleotide into specific probes ofthe array or plate.

For screening, the chip, plate, or array is contacted with a nucleicacid sample (e.g., genomic DNA, cRNA, cDNA, or amplified copiesthereof), which is allowed to hybridize under stringent conditions. Thechip, plate, or array is then scanned to determine which targets havehybridized to which probes. The probes are arrayed in known locations soa signal detected at a specific location indicates that its target hashybridized thereto.

Methods for directly synthesizing on or attaching nucleic acid probes tosolid substrates are available in the art. See, e.g., U.S. Pat. Nos.5,837,832 and 5,837,860, both of which are expressly incorporated byreference herein in their entireties. A variety of methods have beenutilized to either permanently or removably attach the probes to thesubstrate. Exemplary methods include: the immobilization of biotinylatednucleic acid molecules to avidin/streptavidin coated supports(Holmstrom, (Anal. Biochem. 209: 278283 (1993)), the direct covalentattachment of short, 5′-phosphorylated primers to chemically modifiedpolystyrene plates (Rasmussen et al., Anal. Biochem. 198: 138-142(1991)), or the precoating of the polystyrene or glass solid phases withpoly-L-Lys or poly L-Lys, Phe, followed by the covalent attachment ofeither amino- or sulfhydryl-modified oligonucleotides using bifunctionalcrosslinking reagents (Running et al., BioTechniques 8: 276 277 (1990);Newton, C. R. et al., Acids Res. 21: 1155-1162 (1993)). When immobilizedonto a substrate, the probes are typically stabilized and therefore canbe used repeatedly.

Hybridization can performed on an immobilized probe that is attached toa solid surface such as silicon, plastic, nitrocellulose, nylon or glassby addition of one or more target molecules. In some embodiments, thetarget nucleic acid can be attached to a solid surface and the probe canbe added to the immobilized target nucleic acids. Numerous substrateand/or matrix materials can be used, including reinforced nitrocellulosemembrane, activated quartz, activated glass, polyvinylidene difluoride(PVDF) membranes, polystyrene, polyacrylamide, poly(vinyl chloride),poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (whichcontain photoreactive species such as nitrenes, carbenes and ketylradicals capable of forming covalent links with target molecules), andcombinations thereof.

The term “hybridization” includes a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction,primer extension, or the enzymatic cleavage of a polynucleotide by aribozyme.

Hybridization reactions can be performed under conditions of different“stringency”. The stringency of a hybridization reaction includes thedifficulty with which any two nucleic acid molecules will hybridize toone another. Under low to medium stringent conditions, nucleic acidmolecules at least 60%, 65%, 70%, 75% identical to each other remainhybridized to each other, whereas molecules with lower percent identitycannot remain hybridized. For detection of single base polymorphisms,higher stringency conditions can be used.

A preferred, non-limiting example of highly stringent hybridizationconditions include hybridization in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 50° C., preferably at 55° C., more preferably at 60° C., and evenmore preferably at 65° C.

When hybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides, the reaction is called “annealing” andthose polynucleotides are described as “complementary”. Adouble-stranded polynucleotide can be “complementary” and/or“homologous” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.

Complementarity” or “homology” (the degree that one polynucleotide isidentical or complementary to another) is quantifiable in terms of theproportion of bases in opposing strands that are expected to hydrogenbond with each other, according to generally accepted base-pairingrules.

Detection/Identification of Genetic Variants in Expressed Polypeptides

Genetic variants present in polypeptides such as ADRB1, ADRB2,cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE),angiotensinogen, angiotensin receptor, renin, Na⁺ channels (such asSCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such asSLC12A3), and/or WNK1 can be detected by use of binding entities such asantibodies. Detection of specific differences in these polypeptides canbe used to evaluate which blood pressure mediation is more effective.

Altered polypeptides can be detected in a selected fluid or tissuesample (e.g., cell scrapings, saliva, hair follicle, blood, skin tissue,or any convenient sample of a subject's nucleic acids). Any availablemethods for detecting polypeptides can be employed. Examples of suchmethods include immunoassay, Western blotting, enzyme-linkedimmunosorbant assays (ELISAs), radioimmunoassay, immunocytochemistry,immunohistochemistry, flow cytometry, immunoprecipitation, one- andtwo-dimensional electrophoresis, mass spectroscopy and/or detection ofenzymatic activity.

Altered polypeptides can be detected by binding entities.

Antibodies and other binding entities can be used to detect geneticvariants present in ADRB1, ADRB2, cytochrome P450 2D6 (CYP2D6),angiotensin converting enzyme (ACE), angiotensinogen, angiotensinreceptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺)chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1polypeptides. Such antibodies and binding entities can be prepared byavailable methods. For example, available amino acid sequences ofnon-variant and genetic variant ADRB1, ADRB2, CYP2D6, angiotensinconverting enzyme (ACE), angiotensinogen, angiotensin receptors, renin,Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻)co-transporters (such as SLC12A3), and/or WNK1, including thoseillustrated herein, can be used to make antibodies and binding entities.Suitable antibodies may include polyclonal, monoclonal, fragments (suchas Fab fragments), single chain antibodies and other forms of specificbinding molecules. Briefly, these polypeptide detection assays caninclude contacting a test sample with an antibody specific to thegenetic variant site in the polypeptide, detecting the presence of acomplex between the antibody and the polypeptide. In some embodiments, asignal from the polypeptide-antibody complex is detected.

Such antibody-based detection methods can any convenientimmuno-detection method such as Western Blot, ELISA, radioimmunoassay,immunocytochemistry, immunohistochemistry, flow cytometry, andimmunoprecipitation.

Antibodies can be used to detect or identify the presence of geneticvariant forms of ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme(ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (suchas SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (suchas SLC12A3), and/or WNK1 polypeptides in a sample. The antibodies arespecific for sites of genetic variations, and exhibit substantially no(or significantly less) binding to similar polypeptides that do not havethe same genetic variation(s).

Generally speaking, such antibodies can be employed in any type ofimmunoassay, so long as the genetic variations in the polypeptides arereliably identified. This includes both the two-site sandwich assay andthe single site immunoassay of the non-competitive type, as well as intraditional competitive binding assays.

For example, in a typical forward sandwich assay, unlabeled antibody isimmobilized on a solid substrate, e.g., within microtiter plate wells,and the sample to be tested is brought into contact with the boundmolecule. After a suitable period of incubation, for a period of timesufficient to allow formation of an antibody-antigen binary complex, asecond antibody, labeled with a reporter molecule capable of emitting orinducing a detectable signal, is then added and incubation is continuedallowing sufficient time for binding with the antigen at a differentsite and the formation of a ternary complex of antibody-antigen-labeledantibody. Any unreacted material is washed away, and the presence of theantigen is determined by observation of a signal, which may bequantified by comparison with a control sample containing known amountsof antigen.

Variations on the forward sandwich assay include the simultaneous assay,in which both sample and antibody are added simultaneously to the boundantibody, or a reverse sandwich assay in which the labeled antibody andsample to be tested are first combined, incubated and added to theunlabeled surface bound antibody. These techniques are well known tothose skilled in the art, and the possibility of minor variations willbe readily apparent. As used herein, “sandwich assay” is intended toencompass all variations on the basic two-site technique.

For the sandwich assays, the only limiting factor is that bothantibodies have different binding specificities for the genetic variantpolypeptide. Thus, a number of possible combinations are possible. Forexample, a primary antibody can bind specifically to the variant epitopeof one of the variant polypeptides. A secondary antibody can bind to adifferent site on the genetic variant polypeptide. As a more specificexample, in a typical forward sandwich assay, a primary antibody iseither covalently or passively bound to a solid support. The solidsurface is usually glass or a polymer, the most commonly used polymersbeing cellulose, polyacrylamide, nylon, polystyrene, polyvinylchlorideor polypropylene. The solid supports may be in the form of tubes, beads,discs or microplates, or any other surfaces suitable for conducting animmunoassay.

Conventional antibody binding processes can be employed. Followingbinding, the solid phase-antibody complex is washed in preparation forthe test sample. An aliquot of the test sample is then added to thesolid phase complex and incubated at about 25° C. for a period of timesufficient to allow binding of any genetic variant polypeptides presentto the antibody. The primary antibody can bind specifically to the siteof the genetic variant (e.g., the region of a variant amino acid and/orthe structural changes associated therewith), but not to similarpolypeptides that have no such genetic variant. After washing offunbound antibodies, the second antibody is then added to the solid phasecomplex and incubated at 25° C. for an additional period of timesufficient to allow the second antibody to bind to the primaryantibody-antigen solid phase complex (e.g., to a different site on thegenetic variant polypeptide than is bound by the primary antibody). Thesecond antibody may be linked to a reporter molecule, the visible signalof which is used to indicate the binding of the second antibody to anyantigen in the sample.

As used herein, a “reporter molecule” or “label” is a molecule thatprovides an analytically detectable signal, allowing the detection ofantigen-bound antibody. In some embodiments, detection is preferably atleast relatively quantifiable, to allow determination of the amount ofantigen in the sample, this may be calculated in absolute terms, or maybe done in comparison with a standard (or series of standards)containing a known normal level of antigen. The term “label” is usedinterchangeably with “reporter molecule.”

Many commonly used reporter molecules in this type of assay are eitherenzymes or fluorophores. In the case of an enzyme immunoassay an enzymeis conjugated to the second antibody, often by means of glutaraldehydeor periodate. As will be readily recognized, however, a wide variety ofdifferent conjugation techniques exist, which are well known to theskilled artisan. Commonly used enzymes include horseradish peroxidase,glucose oxidase, beta-galactosidase and alkaline phosphatase, amongothers. The substrates to be used with the specific enzymes aregenerally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. For example,p-nitrophenyl phosphate is suitable for use with alkaline phosphataseconjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidineare commonly used. It is also possible to employ fluorogenic substrates,which yield a fluorescent product rather than the chromogenic substratesnoted above. In all cases, the enzyme-labeled antibody is added to thefirst antibody-antigen complex and allowed to bind to the complex, andthen the excess reagent is washed away. A solution containing theappropriate substrate is then added to the tertiary complex ofantibody-antigen-labeled antibody. The substrate reacts with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantified, usually spectrophotometrically, to give anevaluation of the amount of antigen that is present in the serum sample.

Additionally, fluorescent compounds, such as fluorescein or rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorophore-labeled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.As in the enzyme immunoassay (EIA), the fluorescent-labeled antibody isallowed to bind to the first antibody-tagged polypeptide complex. Afterwashing the unbound reagent, the remaining ternary complex is thenexposed to light of the appropriate wavelength, and the fluorescenceobserved indicates the presence of the antigen.

Immunofluorescence and EIA techniques are both very well established inthe art and are particularly preferred for the present method. However,other reporter molecules, such as radioisotopes, chemiluminescent orbioluminescent molecules may also be employed. It will be readilyapparent to the skilled artisan how to vary the procedure to suit therequired use.

In another embodiment, the sample to be tested may be used in a singlesite immunoassay wherein it is adhered to a solid substrate eithercovalently or non-covalently. An unlabeled antibody is brought intocontact with the sample bound on the solid substrate. After a suitableperiod of incubation, for a period of time sufficient to allow formationof an antibody-antigen binary complex a second antibody, labeled with areporter molecule capable of inducing a detectable signal, is then addedand incubation is continued allowing sufficient time for the formationof a ternary complex of antigen-antibody-labeled antibody. For thesingle site immunoassay, the second antibody may be a general antibody(i.e., xenogeneic antibody to immunoglobulin, particularly anti-(IgM andIgG) linked to a reporter molecule) that is capable of binding anantibody that is specific for the genetic variant polypeptide ofinterest.

Kits

Another aspect of the invention is one or more kits for evaluating bloodpressure from a test sample provided by, or obtained from, a subject.

The kits can include any reagents, components and instructions usefulfor testing, assaying, detecting, identifying, and/or determiningwhether genetic variations are present in ADRB1, ADRB2, CYP2D6,angiotensin converting enzyme (ACE), angiotensinogen, angiotensinreceptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺)chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1polypeptides or nucleic acids that can be present in the test samples.

The kits can include reagents, components and instructions fordetecting, identifying, and/or quantifying such polypeptides or nucleicacids. For example, the kits may include primers, probes, labels,enzymes and/or other components for detecting, and/or identifyinggenetic variations in such polypeptides or nucleic acids.

In other embodiments, the kits may include one or more antibodypreparations that selectively bind to genetic variant ADRB1, ADRB2,CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen,angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin,sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/orWNK1 polypeptides, and a detection means for detecting an antibodycomplex that can form (e.g., a label or reporter molecule that is eitherbound to an antibody or is capable of binding to the antibody).

One type of kit can include components for obtaining a sample from asubject, and instructions for sample collection. For example, such asample collection kit can include one or more containers for samplecollection such as one or more vials, test tubes, or receptacles. Thesample collection containers can include a solution for stabilizingsamples placed in the containers. Such a stabilizing solution caninclude protease inhibitors, nuclease inhibitors, DNase inhibitors,RNase inhibitors, chelators, denaturants, salts, salts, and/or buffers.The sample collection kit can also include components for samplecollection such as swabs, droppers, syringes, needles, scalpels, and/orcatheters. The instructions can include steps for sample collection,storage of the sample, and submission of the sample.

The kits can include one or more probes and/or primers each capable ofspecifically binding to a nucleic acid segment of at least 13, 14, 15,16, 17, 18, 19, 20, or 25 nucleotides. In some embodiments, probesand/or primers are each capable of specifically binding to a nucleicacid segment of 15-30, 15-40, 15-50 nucleotides, or any number ofnucleotides between 13-50 nucleotides, in a target DNA or RNA. Theprobes may be part of an array, microarray, microchip, assay plate, ornanochip. Alternatively, the probes or primers may be packagedseparately and/or individually. In some embodiments, the probes orprimers may be detectably labeled. For example, labels can be includedon immobilized probes, where the label signals are quenched untilhybridization occurs and then, upon hybridization, the label emits adetectable signal. Alternatively, one or more labels can be included inthe kit that can bind to a hybridized complex between a probe and atarget DNA or RNA.

Additional reagents can be included in the kits. For example, the kitsmay also contain reagents for detecting or identifying a genetic variantin an ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE),angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such asSCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such asSLC12A3), and/or WNK1 nucleic acid in a test sample. Such reagents caninclude reagents for isolating, storing and detecting nucleic acids. Forexample, the kits can include reagents and enzymes for nucleic acidamplification, primer extension, RNA reverse transcription, sequencing,restriction enzyme cleavage, and/or separation of nucleic acids. Thekits may also include reagents such as solutions for stabilizing nucleicacids, solutions for purifying nucleic acids, nucleotide triphosphates,buffers, and/or other reagents that can be used in in a test tissuesample.

Preservatives and/or antimicrobial agents can be included to stabilizereagents and prevent contamination, such as, for example, paraben,chlorobutanol, phenol sorbic acid, and the like.

It may also be desirable to include agents such as solvents for nucleicacids, reducing agents (e.g., beta-mercaptoethanol), stabilizingreagents (e.g., reagents for inhibiting nucleases, ribonucleases,disrupting tissues, precipitating nucleic acids, and the like).

In further embodiments, the kits can include a computer program productfor use in conjunction with a computer system and the methods describedherein. A computer program mechanism can be embedded in the computerprogram product. The computer program product can, for example, be adevice with a computer program mechanism encoded thereon, where thecomputer program mechanism may be loaded into the memory of a computerand cause the computer to carry out at least one step of a method forassessing the malignant/benign status of a test thyroid tissue sample.For example, the device can be a computer readable storage medium, aflash memory, a compact disc (CD), a digital versatile disc, digitalvideo disc, or an article of manufacture that tangibly includes one ormore computer programs and memory storage. In some embodiments, thecomputer program product can be a computer readable storage medium. Insuch kits, the computer program mechanism can include instructions fordetermining, detecting, and/or identifying a genetic variant in anADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE),angiotensinogen, angiotensin receptors, renin, Na⁺ channel (such asSCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporter (such asSLC12A3), and/or WNK1 nucleic acid or polypeptide in a test sample.

In other embodiments, the kits can include a system, such as a computer,having a central processing unit and a memory coupled to the centralprocessing unit. The memory may store instructions for determining,detecting, and/or identifying a genetic variant in an ADRB1, ADRB2,CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen,angiotensin receptors, renin, Na⁺ channel (such as SCNN1A), adducin,sodium (Na⁺) chloride (Cl⁻) co-transporter (such as SLC12A3), and/orWNK1 nucleic acid or polypeptide in a test sample. The memory can alsostore therapeutic options for different genotyping results.

The kits can also include one or more therapeutic agents, for example,any blood pressure medications described herein.

Definitions

Some definitions are provided below; other definitions are provided inthe other sections of the applications.

As used herein, “obtaining a test sample” involves removing a sample oftissue from a patient, receiving a sample of tissue from a patient,receiving a patient's tissue sample from a physician, receiving apatient's tissue sample via mail delivery and/or removing a patient'stissue sample from a storage apparatus (e.g., a refrigerator or freezer)or a facility. Thus, obtaining a test sample can involve removal orreceipt of the test sample directly from the patient, but obtaining atest sample can also include receipt of a test sample indirectly from amedical worker, from a storage apparatus/facility, from a mail deliveryservice after transportation from a medical facility, and anycombination thereof. The test sample can therefore originate in onelocation, and be transported to another location where it is receivedand tested. Any of these activities or combinations of activitiesinvolves “obtaining a test sample.”

As used herein a probe refers to a single DNA or RNA molecule (a nucleicacid oligomer) or a collection of nucleic acid molecules (nucleic acidoligomers) where the DNA molecules have at least one segment with asequence that is complementary to a region of a target nucleic acid. Theprobe can hybridize with the target nucleic acid under stringentconditions. In some cases, the probe can hybridize with the targetnucleic acid under highly stringent conditions. The probe is notidentical to naturally available nucleic acids because has additionalcomponents such as one or more labels, one or more (engineered)restriction sites, one or more molecular barcodes, one or more tags foridentification or retrieval of the probe (e.g., with or without thetarget hybridized thereto). In some instances the probe is attached to asolid surface such as a chip, an array, a bead, or other surface.

As used herein a primer contains a region that is designed to hybridizeto a targeted locus (e.g., a targeted polymorphic locus or anonpolymorphic locus). The primer and may contain a priming sequencedesigned to allow PCR amplification. The primer can have at least onesegment with a sequence that is complementary to a region of a targetnucleic acid. The primer can hybridize with the target nucleic acidunder stringent conditions. In some cases, the primer can hybridize withthe target nucleic acid under highly stringent conditions. The primer isnot identical to naturally available nucleic acids because hasadditional components such as a molecular barcode, a tag, an engineeredrestriction site, or a combination thereof. A primer may contain arandom region that differs for each individual molecule. The terms “testprimer” and “candidate primer” are not meant to be limiting and mayrefer to any of the primers disclosed herein.

As used herein a “binding entity” is a molecule or molecular complexthat can recognize and bind to selected target molecules. Such bindingentities can be antibodies or any molecule that has a binding domain fora target molecule.

A number of proteins can serve as protein scaffolds to which bindingdomains for targets can be attached and thereby form a suitable bindingentity. The binding domains bind or interact with the targets of theinvention while the protein scaffold merely holds and stabilizes thebinding domains so that they can bind. A number of protein scaffolds canbe used. For example, phage capsid proteins can be used. See Review inClackson & Wells, Trends Biotechnol. 12:173-184 (1994). Phage capsidproteins have been used as scaffolds for displaying random peptidesequences, including bovine pancreatic trypsin inhibitor (Roberts etal., PNAS 89:2429-2433 (1992)), human growth hormone (Lowman et al.,Biochemistry 30:10832-10838 (1991)), Venturini et al., Protein PeptideLetters 1:70-75 (1994)), and the IgG binding domain of Streptococcus(O'Neil et al., Techniques in Protein Chemistry V (Crabb, L. ed.) pp.517-524, Academic Press, San Diego (1994)). These scaffolds havedisplayed a single randomized loop or region that can be modified toinclude binding domains for selected targets.

Researchers have also used the small 74 amino acid α-amylase inhibitorTendamistat as a presentation scaffold on the filamentous phage M13.McConnell, S. J & Hoess, R. H., J. Mol. Biol. 250:460-470 (1995).Tendamistat is a β-sheet protein from Streptomyces tendae. It has anumber of features that make it an attractive scaffold for bindingpeptides, including its small size, stability, and the availability ofhigh resolution NMR and X-ray structural data. The overall topology ofTendamistat is similar to that of an immunoglobulin domain, with twoβ-sheets connected by a series of loops. In contrast to immunoglobulindomains, the β-sheets of Tendamistat are held together with two ratherthan one disulfide bond, accounting for the considerable stability ofthe protein. The loops of Tendamistat can serve a similar function tothe CDR loops found in immunoglobulins and can be easily randomized byin vitro mutagenesis. Tendamistat is derived from Streptomyces tendaeand may be antigenic in humans. Hence, binding entities that employTendamistat are preferably employed in vitro.

Fibronectin type III domain has also been used as a protein scaffold towhich binding entities can be attached. Fibronectin type III is part ofa large subfamily (Fn3 family or s-type Ig family) of the immunoglobulinsuperfamily. Sequences, vectors and cloning procedures for using such afibronectin type III domain as a protein scaffold for binding entities(e.g. CDR peptides) are provided, for example, in U.S. PatentApplication Publication 20020019517. See also, Bork, P. & Doolittle, R.F. (1992) Proposed acquisition of an animal protein domain by bacteria.Proc. Natl. Acad. Sci. USA 89, 8990-8994; Jones, E. Y. (1993) Theimmunoglobulin superfamily Curr. Opinion Struct. Biol. 3, 846-852; Bork,P., Hom, L. & Sander, C. (1994) The immunoglobulin fold. Structuralclassification, sequence patterns and common core. J. Mol. Biol. 242,309-320; Campbell, I. D. & Spitzfaden, C. (1994) Building proteins withfibronectin type III modules Structure 2, 233-337; Harpez, Y. & Chothia,C. (1994).

The following non-limiting examples further illustrate aspects of theinvention.

Example 1: Sample Processing

Each patient is given a collection kit consisting of two buccal swabsand two uniquely barcoded tubes (termed A and B swabs) containing aproprietary lysis buffer consisting of 50 mM Tris pH 8.0, 50 mM EDTA, 25mM Sucrose, 100 mM NaCl, and 1% SDS. The patient will use the swab tocollect buccal cells by scraping the inside of their cheek and place theswab in the provided barcoded tube, one swab for each cheek. Once theswab has been placed into the lysis buffer the cells are no longerviable and therefore samples are now considered to be nucleic acids andsafe to be shipped via standard mail. All samples are checked-in. Thebarcodes of the samples are scanned and their arrival in the laboratoryis confirmed.

FIGS. 3A-3B show schematic diagrams illustrating slight variations insample processing. In general, two samples (Swab A and Swab B) aretaken. The Swab A sample is subjected to the process (DNA Extractionthrough Reporting) unless the Swab A sample fails either the DNA Yieldand Purity Assays, Genomic Analysis, or the PCR QA Assay. If suchfailure occurs, then the other sample (Swab B) is subjected to theprocess, as illustrated in FIGS. 3A and/or 3B.

The samples are grouped into sets of 91 and assigned positions in 96sample grids (12×8 grid layout) for DNA extraction. The remaining fivepositions in each grid can be extraction controls (four negativecontrols [H₂O] and one non-human positive). The five controls can beassigned random positions in each grid, giving each grid/plate a unique“plate fingerprint.” The randomly assigned controls prevent possibleplate swaps or 180° rotations as every plate is now identifiable simplyby control positions. All samples are then normalized to a volume of 650ul by addition of the above mentioned lysis buffer. Additionally, 25 ulof proteinase K (ProK) is added and each sample is incubated in a 55° C.oven for a minimum of 4 hours.

Following such incubation, the samples are extracted using a BioSprint96(KingFisher96) Robotic workstation with magnetic-particle DNApurification chemistry to isolate genomic DNA (GenomicDNA) from tissuesamples. This protocol utilizes the chemistry from the eVoMagDNAExtraction KF96 Kit (Verde Labs, Marietta, Ga.) and is run tospecifications provided by the manufacturer.

Following DNA extraction and subsequent desiccation, the DNA isresuspended in HPLC water. Five microliters of each sample is thenaliquoted to assay plates for the first pair of QA assays, both aPicoGreen fluorometric quantification and a spectrophotometric purityestimation. The fluorescence and absorbance data is analyzed for allsamples in the 96 well plate, including the five controls. The positionsof the negative controls is confirmed and accessed for possible platecontamination. The results for the positive control as well as thesamples on the plate are analyzed for quality metrics using a systemsanalysis approach. The outliers are statistically assessed. After thequantification and purity evaluations, QA assay robotic systems are usedto transfer the samples into racks of 96 sample septa sealed plates (toensure there is no evaporative loss) and a fractional volume of eachsample is used to create a daughter plate of the samples at a normalizedconcentration of 5 ng/μl for the PCR QA assays and subsequentgenotyping. The creation of the normalized daughter plate serves twopurposes. First, it allows the immediate storage of the primary stock ofeach sample at −80° C. avoiding the need for unnecessary freeze-thaw ofsamples and the potential contamination risks associated with repeatedaccessing of the stock. Second, it avoids unnecessary waste of the DNAassociated with the use of full concentration stock for the PCRapplications (this −80° C. stock DNA can be used at any time or savedfor future testing).

Any samples that fail any of the QA assays can re-enter the pipeline andbe sorted and re-processed from the B-swab, which is the secondtube/swab in the kit sent to the customer mentioned above. By alwayshaving a backup sample it is not necessary to go back to the customer toask for a re-swab. If the quantity and purity are still insufficientthen whole genome amplification and/or organic re-extraction can beemployed.

Following the passage of the QA thresholds normalized fractions of thesamples are transferred to PCR plates for genotyping. Each sample isanalyzed using three different methodologies, the Sequenom MassArraygenotyping platform, Sanger sequencing using the ABI 3730x1 genomicanalyzer from Applied Biosystems, and classical PCR and gel sizing todetermine insertion/deletion status. The Sequenom MassArray genotypingplatform is used to analyze the following SNP sites: rs1042713,rs1042714, rs1159744, rs12750834, rs1801252, rs1801253, rs2107614,rs227869, rs4244285, rs4961, and rs699. Sanger sequencing is used toanalyze the following SNPs: rs3892097, rs3758581, rs2228586, and rs5186.Finally classical gel sizing is used to determine the insertion/deletionstatus of the rs1799752 SNP.

Example 2: Sequenom MassArray Assay Design and Processing

The Sequenom platform is able to perform genotyping as a twelve-plexassay (testing 12 variable sites in one reaction) in a 96 well formatusing one aliquot of DNA. The AssayDesign software from Sequenom is usedto generate both PCR and single base extension primers using theindividual rs number of each variable site to create the final assaydesign. Table 1 shows examples of primers that can be used to detectvarious single nucleotide polymorphisms.

TABLE 1 Primers for Amplification of Nucleic Acid Variant SegmentsSNP ID 2^(nd) PCRP SEQ ID 1^(st) PCRP SEQ ID rs1042714ACCTTGGATGAGACATGACGATGCCCATGC NO: 36 ACCTTGGATGAGCGCCTTCTTGCTGGCACNO: 37 rs699 ACCTTGGATGCTGTGACAGGATGGAAGACT NO: 38ACCTTGGATGTGGACGTAGGTGTTGAAAGC NO: 39 rs4961ACCTTGGATGTGTTCGTCCACACCTTAGTC NO: 40 ACCTTGGATGACAAGATGGCTGAACTCTGGNO: 41 rs12750834 ACCTTGGATGGGAATCCAGGAGAATAGGTC NO: 42ACCTTGGATGACAGGCTACCTGGCTTTAAC NO: 43 rs1801252ACCTTGGATGGCCTCGTTGCTGCCTCCCG NO: 44 ACCTTGGATGATCACCAGACCCATGCCCGNO: 45 rs1801253 ACCTTGGATGAGCCCTGCGCGCGCACCA NO: 46ACCTTGGATGICAACCCCATCATCTACTGC NO: 47 rs227869ACCTTGGATGCTGACATTGCCACCTGTATC NO: 48 ACCTTGGATCGTAGTGGCACTGGCATATTCNO: 49 rs2107614 ACCTTGGATGGCAACCATCACAGTACTAAG NO: 50ACCTTGGATCCACAACTGGAAGAGTTGAGG NO: 51 rs1529927ACCTTGGATGTGGACCCCATTAACGACATC NO: 52 ACCTTGGATGTCACCTTGGACTCCCACTCNO: 53 rs4244285 ACCTTGGATCCACTTTCCATAAAACCAAGG NO: 54ACCTTGGATGGCAATAATTTTCCCACTATC NO: 55 rs1042713ACCTTGGATGATGAGAGACATGACGATGCC NO: 56 ACCTTGGATGGAACCGCACCGCCTTCTTGNO: 57 rs1159744 ACCTTGGATGGAAACAGTGACAGCCAAATG NO: 58ACCTTGGATGGTTTTTCACTTCCTGAATTTG NO: 59

DNA samples at a concentration of 5 ng/ul undergo a PCR using the abovedesigned PCR primers and the Sequenom iPLEX Gold Reagent kit under theconditions described in Table 2.

TABLE 2 PCR Reaction Mixture Reagent Final Concentration Vol/rxn (uL)Water, HPLC n/a 1.8 10x PCR Buffer with 20 mM MgCl₂ 2 mM MgCl₂ 0.5 25 mMMgCl₂ 2 mM 0.4 25 mM dNTP Mix 500 uM 0.1 0.5 mM Primer Mix 0.1 uM 1 5U/uL PCR Enzyme 1 unit 0.2 Volume 4 10 ng/uL DNA 10 ng/rxn 1 TotalVolume 5

The PCR reaction cycling conditions can be as illustrated in Table 3.

TABLE 3 PCR Reaction Cycling Cycler Program iPlex- PCR Temp (° C.) Time(min) 95 2:00 95 0:30 Repeat 56 0:30 45 72 1:00 Cycles 72 5:00 4 ∞

Directly following PCR amplification, excess primers and deoxynucleotidetriphosphates are removed via a SAP (shrimp alkaline phosphatase)reaction under the conditions described in Table 4.

TABLE 4 PCR Clean-Up Reagent Final Concentration Vol/rxn (uL) Water,HPLC N/A 1.53 SAP Buffer (10x) 0.24x 0.17 5 U/uL PCR Enzyme 1 unit 0.2Volume 2 PCR product 5 Total Volume 7

The Shrimp Alkaline Phosphatase reaction is incubated at 37° C. for 40min, followed by incubation at 85° C. for 5 min. The samples can bestored at 4° C. indefinitely.

After the SAP reaction is completed the samples can be subjected tosingle base extension reactions using the primers described in Table 5,and the conditions described in Table 6 and 7.

TABLE 5 Single Base Extension Primers SEQ ID SNP Sequence NO: rs1042714ACACCTCGTCCCTTT 60 rs699 CTGGCTGCTCCCTGA 61 rs4961 ACTGCTTCCATTCTGCC 62rs12750834 AGTCTCTGTAAGTGCCC 63 rs1801252 GTGCCTCCCGCCAGCGAA 64rs1801253 CGCGCGCAGCAGAGCAGT 65 rs227869 AGCTGTATCTGCTCCATTCA 66rs2107614 TCCTCCAAAAAAAAAGAAAAC 67 rs1529927 GTTACCGACATCCGCATCATTG 68rs4244285 TAAGTAATTTGTTATGGGTTCC 69 rs1042713 GGAGGGGTCCGGCGCATGGCTTC 70rs1159744 CAAATGTTAACAGTATAGAAAATTTTA 71

TABLE 6 Single Base Extension Reaction Conditions Reagents FinalConcentration Vol/rxn(uL) Water, HPLC N/A 0.619 iPlex Gold Buffer0.222x   0.200 iPlex Termination Mix 1x 0.200 iPlex Extend Primer Mixvaries 0.940 iPlex Enzyme 1x 0.041 Volume 2.000 PCR product 7 TotalVolume 9

TABLE 7 Single Base Extension Reaction Cycling conditions Temp (∞ C.)Time (min) 94 0:30 94 0:05 40 cycles 52 0:05 5 cycles ↓ 80 0:05 ↓ 723:00 4 forever

After completion of all of the above reactions, the samples are runthrough resin based clean-up to remove excess salts according tostandard Sequenom protocols. The samples are then spotted onto theSequenom provided SpectroChip using the Sequenom Nanodispenser accordingto manufacturer protocols and subsequently processed on the SequenomMALDI-TOF platform.

A sample results report is provided in Table 7. The two letters for eachpolymorphism type are for the two alleles present in the subjects,illustrating that the subjects are homozygous for some polymorphisms(e.g., subject GCE0104 is homozygous (G/G) for the variable site in thers1042713 polymorphism, but subject GCE0120 is heterozygous (GA) forthat site).

TABLE 8 Results SNP GCE0120 GCE0104 rs1042713 GA GG rs1042714 GC GGrs1159744 AA AT rs12750834 GA GG rs1529927 GG GG rs1801252 AA AArs1801253 GG GG rs2107614 CT TT rs227869 AA AG rs4244285 GG GA rs4961 GGGG rs699 CG GG

Example 3: Sanger Sequencing Primer Design and Workflow

All primers for Sanger sequencing were designed using the free,web-based primer design tool Primer3. 500 base pairs of flankingsequence for each SNP (single nucleotide polymorphism) were entered intoPrimer3. The top primer candidate for each site was chosen and optimizedusing a buffer panel with varying MgCl concentrations and a temperaturegradient to determine the optimal cycling conditions for each primerpair.

TABLE 9 Primers for Sequencing of SNPs Primer Name Sequence SEQ IDPurpose rs3892097_F TTCAGTCCCTCCTGAGCTA NO: 72 SNP rs3892097_RAAGGTGGATGCACAAAGAG NO: 73 SNP rs3758581_F GTGCATCTGTAGCAGTCCTC NO: 74SNP rs3758581_R CCAAACTGGAATCAACAGAA NO: 75 SNP rs2228586_FGAAGTGGTCTCGTCTAGCAA NO: 76 SNP rs2228586_R CAGAGAGAGAGGTCCCATTT NO: 77SNP rs5186_F CCACTCAAACCTTTCAACAA NO: 78 SNP rs5186_RTGGACAGAACAATCTGGAAC NO: 79 SNP

The region encompassing the SNP was amplified from sample nucleic acidsby PCR using optimized individual cycling conditions for each SNP site.Directly after PCR amplification each sample is cleaned up using a sizeexclusion micro-filtration plate from Millipore and entered into theSanger sequencing reaction. Each sample is sequenced in both the forward(3′) and reverse (5′) direction giving double conformation of theallelic state. These forward and reverse sequences from each patient arethen aligned to the human reference sequence using the CLC DNA workbenchprogram creating an alignment file from which the allele call is madeand added to the final SNP call report.

FIG. 6 illustrates the results from one such alignment.

Example 4: Gel Sizing Primer Design and Workflow

To accurately call the insertion/deletion status for site rs1799752, PCRamplification of sample nucleic acids is performed followed by gelelectrophoresis. The PCR primers for this site were also designed andoptimized using Primer3 and the above mentioned buffer and temperaturegradient. The following primer sequences and PCR conditions wereultimately chosen:

TABLE 10 Primer Sequences for PCR of rs1799752 Insertion/DeletionPrimer Name Sequence SEQ ID Purpose rs1799752_F-2 CCCATTTCTCTAGACCTGCTNO: 80 INDEL rs1799752_R-2 GGGATGGTGTCTCGTACATA NO: 81 INDEL

Following PCR amplification, each sample is loaded into its own well ofa 2% agarose gel and run at 150 mV for approximately 45 min and stainedin a bath of GelRed for 2 hours prior to imaging with UV light. Theresulting image is used to score the presence or absence of a 288 bp ALUvisually by examining the gel for either the higher molecular weightband (indicating the presence of the 288 bp ALU), the lower molecularweight band (indicating the absence of the 288 bp ALU) or both(indicating a heterozygous state. A sample image of the gel is providedin FIG. 7.

Example 5: Genotyping Reports

Once all tests are performed a report is generated containing allresults for each tested patient. One example of a report for subjectsGCE0120 and GCE0104, is shown below. The two letters for eachpolymorphism type are for the two alleles present in the subjects,illustrating that the subjects are homozygous whereas other subjects areheterozygous for the variable site of each polymorphism.

TABLE 11 Results from Analysis of Polymorphisms Polymorphism typeGCE0120 GCE0104 Sequenom Results rs1042713 GA GG rs1042714 GC GGrs1159744 CG CG rs12750834 GA GG rs1529927 GG GG rs1801252 AA AArs1801253 GG GG rs2107614 CT TT rs227869 AA AG rs4244285 GG GA rs4961 GGGG rs699 TT TT Sanger Sequencing Results rs3892097 CC CC rs3758581 GG GGrs3758580 CC CT rs2228586 TT TT rs5186 AC AA Gel Results rs1799752 +/++/−

Example 6: Clinical Study Protocol

Clinical Protocol Summary

Study Title: Assessment of the Relationship between Genes that EncodeProteins Important in Blood Pressure Regulation and Blood PressureTherapy in Patients with Hypertension. Study The GeneticurePharmacogenetic Testing Kit. The kit Device: contains two buccal swabswith two buffer solution vials to stabilize DNA. These buccal swabs areused for DNA collection which is then extracted for analysis of genesimportant in high blood pressure. Target The Geneticure PharmacogeneticTesting Kit is a Indication pharmacogenomic treatment decision supportproduct that for Use: tests for clinically important genetic variantsaffecting a patient's response to antihypertensive medications. StudyThis is a post-hoc association study of patients who have Design: beendiagnosed with high blood pressure and have been stable on medicationtreatment for at least 6 months. Study To be enrolled in this study,subjects must meet ALL of Population: the inclusion criteria and NONE ofthe exclusion criteria: Inclusion Criteria 1. Subject is able andwilling to provide informed consent 2. Subject is ≥30 and ≤70 years ofage 3. Subject with diagnosis of Hypertension for a minimum of 1 year 4.Subject has been on the same class/classes of blood pressure medicationfor a minimum of 6 months. Note: A change in dosage, frequency, orspecific medication is acceptable as long as there have been no changesto the class/classes of medications prescribed. 5. Subject with a BodyMass Index (BMI) ≥19 and ≤35 6. Subject is currently prescribed andtaking one of the following classes of medications alone or incombination with each other or a Ca+ channel blocker. Diuretics ACEInhibitors Angiotensin Receptor Blocker (ARB) Beta-blockers ExclusionCriteria 1. Subject has clinically significant kidney disease asdetermined by the investigator. 2. Subject has clinically significantcardiac disease as determined by the investigator. 3. Subject hasclinically significant vascular disease as determined by theinvestigator. 4. Subject has a diagnosis of secondary hypertension or isexperiencing a complication of pregnancy. 5. Subject is currentlyprescribed and taking any additional class of medication(s) for highblood pressure not included in the list above, with the exception of aCa+ channel blocker. 6. Subject has Systolic BP >190 or DiastolicBP >120 documented within the six months prior to visit. 7. Subject hasa regular alcohol intake of greater than 21 units per week in the past 6months 8. Subject has smoked greater than two packs of cigarettes(total) or equivalent nicotine intake in the past 6 months. 9. Subjecthas an anticipated survival less than 12 months. 10. Any other reasonthat the subject is inappropriate for study enrollment in the opinion ofthe Investigator. Primary To assess the relationship between the drugtherapy Study class/combination of therapy classes that resulted in theObjective: best blood pressure control for a patient vs. what theGeneticure high blood pressure panel would have predicted. Secondary Toassess the clinical time to achieve optimal blood Study pressuretreatment. Objectives: To assess the number of office visits required toachieve optimal blood pressure treatment.

1 Introduction

Hypertension (high blood pressure) is one of the most importantpreventable contributors to disease and death in the United States andrepresents the most common condition seen in the primary care setting(The sixth report of the Joint National Committee on prevention,detection, evaluation, and treatment of high blood pressure. Arch InternMed. 157(21):2413-2446 (1997); Chobanian et al. JAMA 289(19):2560-2572(2003)). According to the American Heart Association, approximately 78million adults (1 in 3) living in the United States have hypertensionwith more than 5 million new diagnoses made each year (American HeartAssociation. Heart and Stroke Statistics—2004 Update. Paper presentedat: Dallas: American Heart Association (2004); Roger et al. Circulation.125(1):e2-e220 (2012)). Of these individuals, 82% are aware they haveit, 75% are currently being treated for it, but only 52% have theirblood pressure under control. Thus, about 48% of individuals withhypertension do not have adequate blood pressure control. Hypertensionis known to lead to myocardial infarction (heart attack), stroke, renalfailure, and death if not detected early and treated appropriately. Infact, in 2009, high blood pressure was listed as a primary orcontributing cause of death in 350,000 of the ˜2.4 million U.S. deaths(14% of all deaths). From 1999-2009 the number of deaths attributable tohypertension increased by 44%.

Refractory (or resistant) hypertension is defined as blood pressure thatremains above clinical guideline goals in spite of concurrent use ofthree antihypertensive agents of different classes (Akpunonu et al.,Disease-a-month: DM. October 1996; 42(10):609-722). Critically,refractory hypertension is noted in approximately 25-30% of allindividuals being treated for hypertension. Refractory hypertension is acommon clinical problem which contributes to the high levels ofmorbidity and mortality. The inability to gain control of blood pressurein these individuals may be related to the pharmacogenetics of theindividual coupled with the specific classes of drugs and/or combinationof classes chosen for that individual (Calhoun et al. Circulation117(25):e510-526 (2008); Johnson & Turner, Curr Opin Mol Ther7(3):218-225 (2005)). In 2009, the direct and indirect economic burdenon the United States health care system associated with hypertension wasestimated at $51 billion. With the advent of improved diagnostictechniques, increased rates of health care utilization and screening,and the increasing age of the population, a continual upward trend inthis expenditure is expected.

Globally, nearly 1 billion individuals have been diagnosed withhypertension with an estimate of an additional 400 million living withundiagnosed hypertension. Hypertension is the leading cause of prematuredeath and the leading cause of cardiovascular disease worldwide. Similarto the continued upward trend in prevalence as seen in the UnitedStates, it is estimated that in 2025 approximately 1.56 billion adultswill be living with hypertension. Because nearly two-thirds of thepeople living with hypertension worldwide reside in developingcountries, providing optimal treatment at the lowest cost is criticallyimportant.

Unfortunately, despite a significant impulse in the medical community tomove towards an “individualized medicine” approach to patient centeredtreatment, the current clinical treatment strategy is based on a setalgorithm which does not take into account individual patientdifferences. Rather, physicians are guided to choose a drug (one out ofmany options) in a given class of drugs and use that specific drug as a“first line therapy” (typically initiating with the diuretic class) andtitrate that specific drug of choice to therapeutic dosage regardless ofefficacy (Chobanian et al. JAMA 289(19):2560-2572 (2003)). It is onlyafter a prolonged course of treatment with that specific class of drugthat clinical efficacy is determined (typically three months). At thisstage, if clinical guideline goals for blood pressure have not been met,it is often recommended that the patient remain on the “first linetherapy” whilst an additional drug from a different class of drugs(typically an Angiotensin converting enzyme inhibitor (ACE inhibitor) orAngiotensin II receptor blocker (ARB)) is added to the pharmacologicregimen. Again, this drug is titrated to recommended therapeutic dosageand another prolonged course of treatment is initiated before clinicalefficacy is determined (an additional three months—six months sinceinitiation of treatment). If at this point, clinical guideline goals forblood pressure have not been met, a third drug from a third class ofdrugs (typically a beta-blocker) is added and the process is repeated(another three months—nine months from initiation of treatment).Further, if clinical guideline goals have continued to be elusive, thediagnosis of refractory hypertension is added and the process isreinitiated with a different combination of drugs, different classes ofdrugs, different drug options within a given class of drugs, differentdosages, or all of the above. Thus, from the time of initial diagnosisand the start of treatment to the point in which blood pressure isadequately controlled may take anywhere from three months to well overone year. This trial-and-error standard of care is clearly not optimal.

The blood pressure panel created by Geneticure has been created tocomprehensively assess fourteen common genetic variants in the cardiac,vascular, and renal systems that can improve therapeutic guidance forthe clinician based on known functional alterations of the proteinthrough these genetic changes, as well as demonstrated effects ofcertain drug classes on these various genotypes. Based on thisinformation, a clinician can guide therapy with knowledge specific totheir patient, rather than “trial-and-error” based on population dataand using drugs with least side effects initially.

1.1 Investigational Device: Geneticure Pharmacogenetic Testing Kit

The Geneticure pharmacogenetic testing kit contains two buccal swabswith two buffer solution vials to stabilize DNA. These buccal swabs areused for DNA collection which is then extracted for analysis of genesimportant in high blood pressure.

The Geneticure Pharmacogenetic Testing Kit is a pharmacogenomictreatment decision support product that tests for clinically importantgenetic variants affecting a patient's response to antihypertensivemedications.

1.2 Genetic Analysis

Each sample can be analyzed for fourteen common genetic variants using 3different methodologies, the Sequenom MassArray genotyping platform,Sanger sequencing using the ABI 3730x1 genomic analyzer from AppliedBiosystems, and classical PCR and gel sizing to determineinsertion/deletion status (see, FIGS. 3A-3B).

2 Methodology

2.1 Study Design and Protocol Overview

This is a post-hoc association study of patients who have been diagnosedwith high blood pressure and have been stable on medication treatmentfor at least 6 months. The purpose of this study is to evaluate therelationship between optimal medication therapy (or the therapy that hasresulted in the most stable blood pressure for that particular patient)and the predicted optimal medication therapy based on a patient'sgenetic profile.

Chart reviews for the patient's history of antihypertensive therapy canbe coupled with buccal swabs and blood pressure readings collected fromeligible patients who have provided informed consent. The swab can beanalyzed for fourteen genetic variants that are associated withantihypertensive therapy response (efficacy, side-effects).

2.2 Study Objective

To assess the relationship between the drug therapy class/combination oftherapy classes that resulted in the best blood pressure control for apatient vs. what the Geneticure high blood pressure panel would havepredicted.

2.3 Secondary Objectives

The secondary objectives are as follows:

-   -   To assess the clinical time to achieve optimal blood pressure        treatment.    -   To assess the number of office visits required to achieve        optimal blood pressure treatment.

3 Investigational Study Center

This study will be conducted at up to 5 study centers within the UnitedStates that have adequate resources for trial responsibilities.

4 Study Population

To be enrolled in this study, subjects must meet ALL of the inclusioncriteria and NONE of the exclusion criteria:

4.1 Inclusion Criteria

-   -   1. Subject is able and willing to provide informed consent    -   2. Subject is ≥30 and ≤70 years of age    -   3. Subject with diagnosis of Hypertension for a minimum of 1        year    -   4. Subject has been on the same class/classes of blood pressure        medication for a minimum of 6 months. Note: A change in dosage,        frequency, or specific medication is acceptable as long as there        have been no changes to the class/classes of medications        prescribed.    -   5. Subject with a Body Mass Index (BMI) ≥19 and ≤35    -   6. Subject is currently prescribed and taking one of the        following classes of medications alone or in combination with        each other or a Ca⁺ channel blocker.        -   Diuretics        -   ACE Inhibitors        -   Angiotensin Receptor Blocker (ARB)        -   Beta-blockers

4.2 Exclusion Criteria

-   -   1. Subject has clinically significant kidney disease as        determined by the investigator.    -   2. Subject has clinically significant cardiac disease as        determined by the investigator.    -   3. Subject has clinically significant vascular disease as        determined by the investigator.    -   4. Subject has a diagnosis of secondary hypertension or is        experiencing a complication of pregnancy.    -   5. Subject is currently prescribed and taking any additional        class of medication(s) for high blood pressure not included in        the list above, with the exception of a Ca+ channel blocker.    -   6. Subject has Systolic BP>190 or Diastolic BP>120 documented        within the six months prior to visit.    -   7. Subject has a regular alcohol intake of greater than 21 units        per week in the past 6 months    -   8. Subject has smoked greater than two packs of cigarettes        (total) or equivalent nicotine intake in the past 6 months.    -   9. Subject has an anticipated survival less than 12 months.    -   10. Any other reason that the subject is inappropriate for study        enrollment in the opinion of the Investigator.

5 Informed Consent

The investigator will prepare an informed consent form in accordancewith this study protocol and all regulatory requirements (21 CFR Part50) using the template informed consent form provided by the sponsor.The informed consent form must be submitted to the IRB and a copy of thefinal IRB-approved consent form must be submitted to the StudyManagement Center prior to the start of the study at thatinvestigational site.

Prior to any study procedures, all subjects must document their consentfor study participation and authorization for use and disclosure ofhealth information by signing the IRB-approved Informed Consent Form. Aspart of the consent process, the subject will have the opportunity toask questions of, and receive answers from the personnel conducting thestudy.

The investigator will notify the Study Management Center and the IRBwithin 5 working days if device use occurs without subject informedconsent.

6 Study Assessments and Data Management

6.1 Screening

-   -   Identify Potential Study Subjects. Refer to the Inclusion and        Exclusion Criteria sections of this protocol for a complete list        of eligibility criteria.    -   Obtain Written Informed Consent. Each potential study        participant must be given time to review the IRB-approved        informed consent form, have his/her questions answered to their        satisfaction and sign the form prior to any study procedures        being performed. A subject will be given a copy of the informed        consent form.    -   Review Inclusion/Exclusion Criteria. The investigator and/or        designee will review all criteria to determine if the subject is        eligible for enrollment. Eligibility of all subjects must        personally be confirmed by the Investigator and will be        documented on the CRF.

6.2 Enrollment

-   -   Assign Identification Number to Eligible Subjects. See Protocol        section 6.3.    -   Record Demographics, Antihypertensive Medical History and        current Blood Pressure. Data will be documented in the source        document and recorded on the CRF, including but not limited to        the following:        -   Age        -   Height        -   Weight        -   Race        -   Ethnicity        -   Length of Hypertension diagnosis        -   Previously and currently prescribed antihypertensive            medications        -   Blood pressure measurements

6.3 Specimen Collection

-   -   Collect Buccal Specimen.        -   Using the collection kit consisting of two buccal swabs and            two uniquely barcoded tubes the investigator or designee            will remove the first buccal brush and scrape the brush end            across a Subject's right cheek repeatedly (for five            seconds). The investigator/designee will place the brush end            over the open buffer vial and press the opposite end of the            swab stick to release the brush into the buffer and then            close the vial. The process can be repeated on the left            cheek. Each of the right and left cheek vial numbers must be            recorded on the CRF and accountability log as right (R), or            left (L).    -   Adverse Event Recording    -   Perform Product Accountability

6.4 Subject Numbering

Subjects meeting the criteria for enrollment (and their specimens) canbe identified by unique numbers that can be assigned sequentially byorder of enrollment. The pre-assigned investigational site number can beprefixed to the identification number and separated by a hyphen (e.g.,site 01 would number their subjects sequentially as 01-001, 01-002,01-003, etc.). Throughout the descriptions within the protocol the Aswab will be referring to the swab that has originated from the rightcheek, while the B swab will be that that has originated from the leftcheek. To further clarify. Subject 01-001 can be given two barcodedtubes. These barcode numbers can be recorded for each patient. These canalso be recorded as originating from the right cheek (A) or left cheek(B).

At no time should any study paperwork or specimens be marked with thesubject's name or any other traceable identifier except for the informedconsent form, which is signed by the subject and kept at the site. At notime should the original (signed) or a copy of this form be collected bythe Sponsor or its representative.

6.5 Subject Completion and Withdrawal

Once subjects undergo the sampling procedure, their study participationis complete. There are no follow-up visits. Subjects will be instructedto notify the Investigator if they experience any symptoms orcomplications from the sampling procedure.

Subjects are free to withdraw consent and discontinue participation inthe study at any time. A subject's participation in the study may bediscontinued at any time at the discretion of the Investigator orGeneticure. The following may be justifiable reasons for theInvestigator or Geneticure to remove a subject from the study:

-   -   The subject was erroneously included in the study or was found        to have an exclusion criterion.    -   The subject was uncooperative.    -   The subject experienced an AE/SAE during the sample collection        procedure that is considered intolerable by the subject or        Investigator.

To the extent possible, safety data will be collected on subjects whodiscontinue participation in the study due to safety reasons.

The following may be justifiable reasons for the Investigator orGeneticure to remove a specimen from the study:

-   -   Sample is determined to be of poor or inadequate quality for        analysis (e.g., contamination, insufficient material for        analysis).    -   The sample was erroneously included in the study.    -   The specimen was not collected or processed per protocol        procedures.

6.6 Concomitant Medications/Treatment/Procedures

This study protocol does not require change to any existing treatmentsor those prescribed during the course of the study by the Investigatoror any other provider whom the subject sees for any medical reason.Outside of eligibility screening, there are no clinical evaluations aspart of this study.

6.7 Data Management

The Investigator is responsible to ensure the accuracy, completeness,and timeliness of reported data.

All data will be sent to Geneticure who will enter it into the studydatabase using a secure, protected Excel spreadsheet. The database willbe validated prior to use in the study. All required data will berecorded on CRFs or paper facsimiles. Data collected within the CRFswill be supported by source documents as appropriate and may be updatedto reflect the latest observations on the subjects participating in thestudy. Corrections to the source documentation can be made in a mannerthat does not obscure the original entry and will be dated and initialedby the Investigator or assigned designee. It is important for data entryto occur in a timely manner, therefore, data collected on sourcedocuments should be transferred into CRFs as soon as possible followingstudy visits.

Study subject data can be reviewed at the investigational site bymonitors at regular intervals throughout the study. Information on theCRFs can be compared to information originally recorded on sourcedocuments related to the study (i.e. professional notes, study-specificworksheets, etc.)

7 Genomic Core Laboratory

The subjects' cheek vials will be sent to the Geneticure processingcenter. The vials will then be batched and sent to the Genomic Corelaboratory for DNA extraction and genetic analysis. Following analysis,results will be sent to Geneticure for statistical analysis and DNA willbe destroyed.

A protocol for the extraction and analysis will be followed to ensureconsistency and objectivity.

8 Adverse Events

The procedures outlined in this protocol do not involve significant riskto subject safety. Subjects will be provided the investigator's contactinformation and will be instructed to notify the investigator of anyadverse events they experience during or secondary to the samplecollection procedures.

8.1 Definitions

8.1.1 Adverse Event

-   -   For the purposes of this study, an adverse event is defined as        any undesirable/unusual medical experience that occurs to a        subject in conjunction with the use of the product, whether or        not considered product related, including (but not limited to)        those events that result from the use as stipulated in the        protocol.    -   The following adverse events will not be collected in this        study:        -   Adverse events which, in the opinion of the Investigator,            are unrelated to the swab collection procedure, but rather            related to the subject's underlying medical conditions or            status        -   Adverse events that may be related to the sample collection            procedure but result only in local, mild and transient            discomforts.    -   The Investigator is responsible for documenting all Adverse        Events on the Adverse Event CRF, except for those events noted        above.

8.1.2 Serious Adverse Event

-   -   A Serious adverse event is an adverse event that:        -   led to death,        -   led to serious deterioration in the health of a subject that            -   resulted in a life-threatening illness or injury,            -   resulted in permanent impairment of a body structure or                body function,            -   required inpatient hospitalization or prolongation of                existing hospitalization,            -   resulted in medical or surgical intervention to prevent                permanent impairment to a body structure or a body                function.        -   led to fetal distress, fetal death or a congenital anomaly            or birth defect.

8.2 Event Reporting

Any AE, or SAE experienced by a subject after signing the informedconsent until twenty-four (24) hours following study completion ortermination will be recorded in the progress notes and on the CRF. TheInvestigator and/or designee will continue to monitor the subject withadditional assessments until the AE is considered resolved, stabilized,or is lost to follow up.

A full description of an adverse event, including the nature, date andtime of onset and resolution, determination of seriousness, frequency,severity, treatment, outcome, and relationship to the study will berecorded on the Adverse Event CRF.

SAEs must be reported to RCRI within 48 hours of the Investigator'sfirst knowledge of the event.

9 Statistical Methods

Following is a summary of the Statistical Analysis Plan for the study.The following objectives have been prospectively defined; however, dueto the nature of these data, additional analyses may be conducted oradditional subsets may be identified that are not listed in thisprotocol.

9.1 Sample Size

-   -   Up to 300 subjects may be enrolled at each site. The minimum        number of subjects for meaningful statistical analysis is 100        subjects.

9.2 Data Analysis

-   -   All data will be coded for statistical analysis (i.e. drug        classes and genotypes will be coded numerically). All data will        be analyzed with SPSS v.20. Normality of the data will be        assessed using Levene's test prior to statistical analysis and        any correction for non-normal data distribution will be used.        Descriptive statistics will be computed (average time for blood        pressure control, average number of visits to the clinician for        blood pressure control, age, height, weight, BMI, etc.).    -   Data will be initially analysed following the collection of        samples/data from 100 subjects. This will allow for direction        for power calculations/etc. for future statistical analysis.        Although some of the genes have been analysed individually, no        mean or standard deviation data exists to allow for a true a        priori power calculation. Data will be analysed again after two        months or following 300 subjects for which data has been        collected. Statistical tests will be corrected for the number of        tests run (preservation of alpha).    -   Ordinary least squares regression via univariate modelling will        be used to estimate the magnitude of linearity between drug        class that yielded the best blood pressure control and genetic        profile of the subject. Multiple regression analysis will be        performed to determine the impact of confounding variables        (height, weight, age, race) on blood pressure control. For all        statistical analyses an alpha level of 0.05 will be used to        determine statistical significance.

9.3 Other Statistical Considerations

Justification of Pooling Data Across Centers

There is no need to keep the data from different centers separate fordata collection. Primary reasons for not pooling blood pressure datafrom different centers could include different races (which we arecollecting as a demographic and analyzing as a co-variate in amultiple-regression) and different cultures (i.e. southern vs. northernhabits of diet, exercise, etc.). The study will take race, height,weight, age into account as co-variates in a multiple regression model,but will not be powered to take into account possible geographicinfluences on the data.

Missing Data

All patients with available data will be included in the analyses ofprimary and secondary objectives. Because some of the data was notrecorded as part of a prospective protocol, an unknown amount of datawill be permanently missing. No patients will be contacted to retrievemissing data, and no sensitivity analyses will be performed on missingdata.

10 Risk Analysis

10.1 Device Description

The procedures outlined in this protocol do not involve significant riskto subject safety. Subjects will be provided the investigator's contactinformation and will be instructed to notify the investigator of anyadverse events they experience during or secondary to the specimencollection procedures.

The collection kit includes a small, soft, brush for cheek swabbing anda buffer solution in a small vial, one of each for each cheek, two intotal. Once the swab has been placed into the lysis buffer the cells areno longer viable and therefore samples are now considered to be nucleicacids and safe to be shipped via standard mail.

11 Study Materials

11.1 Handling and Storage

-   -   The Investigator must ensure that the investigational product is        stored in a controlled location with limited access.

11.2 Product Accountability

-   -   The investigator is responsible for investigational product        accountability, reconciliation and record maintenance. The        investigator must maintain investigational product        accountability records throughout the course of the study.    -   Upon completion or termination of the study, all unused product,        together with a copy of the product accountability form will be        returned to Geneticure or its representative.    -   All supplies are to be used only for this protocol and not for        any other purpose.

12 Study Administration

12.1 Subject Confidentiality

All information and data sent to Geneticure, and/or its designeesconcerning subjects and their participation in this study are consideredconfidential by Geneticure and it designees (subcontractors or contractresearch organization). Only authorized Geneticure personnel or approvedcontracted agents of Geneticure will have access to some portions ofthese confidential files and will act in accordance with applicableregulations as required by HIPAA. The IRBs and FDA also have the rightto inspect and copy all records pertinent to this study. All data usedin the reporting of the study will eliminate identifiable reference tothe subjects.

12.2 Investigational Center Qualification

Investigational Center qualification visits or phone calls will beconducted by the Study Management Center prior to acceptance of the siteinto this study. The site qualification visit will be scheduled toinclude time with the Principal Investigator and other study personnelas applicable. Areas of discussion include a review of personneltraining, investigator qualifications, adequacy of potential subjectpool, FDA-regulated study experience, this study's specific requirementsfor procedures, and a review of staffing availability andappropriateness. A written report of the qualification visit will bedrafted by the Study Management Center.

12.3 Site Training

Study-specific training of study personnel is the responsibility of theSponsor or Study Management Center and the Principal Investigator. Studytraining will occur before the first device use. To ensure protocol andregulatory compliance as well as accurate data collection, site trainingwill include a detailed review of the protocol, CRF completion, studyspecific procedures, monitoring logistics, and regulatory requirements.

12.4 Investigator Responsibilities

The investigator is responsible for ensuring that the study is conductedaccording to the investigational plan and all applicable FDAregulations, including reporting and record-keeping requirements, andcontrolling the devices undergoing investigation and HIPAA. In addition,the principal investigator is responsible for ensuring that informedconsent is obtained from each subject prior to participating in thestudy, as well as protecting the rights, safety and welfare ofparticipating subjects. Specific responsibilities are listed in thisinvestigational plan.

Records and reports must remain on file at the investigational site fora minimum of two years after the later of either thecompletion/termination of the investigational study or the date it isdetermined the records are no longer required to support submissions toregulatory authorities. They may be discarded only upon approval fromGeneticure. The Principal Investigator must contact Geneticure beforedestroying any records and reports pertaining to the study to ensurethat they no longer need to be retained. In addition, Geneticure must becontacted if the investigator plans to leave the investigational site toensure that arrangements for a new investigator or records transfer aremade prior to investigator departure.

12.4.1 Records

-   -   Records to be maintained by the investigator in the designated        investigational center's study file include:        -   Investigational plan and all amendments        -   Signed Financial Disclosure        -   IRB approval letter including consent and HIPAA            authorization form(s)        -   IRB Membership list or Letter of Assurance        -   All correspondence relating to the study between the site            and Geneticure, and the Study Management Center        -   CVs and professional licenses for all investigators        -   Site personnel signature and responsibility list        -   Clinical monitor sign-in log        -   Blank set of each version of CRFs        -   Subject Screening/Enrollment log        -   Investigational device accountability log including: date,            quantity, lot numbers of all devices, identification of all            persons the device was used on and final disposition.

The following records are maintained for each subject enrolled in thestudy:

-   -   Signed Consent Form and Authorization for the Use and Disclosure        of Health Information    -   Compete, accurate and current CRFs and DCFs    -   Adverse event reports and any supporting documentation    -   Protocol deviations    -   Complete medical records, including procedure reports, lab        reports, professional notes, etc.

Geneticure reserves the right to secure data clarification andadditional medical documentation on subjects enrolled in this study atany time.

13 Abbreviations

-   -   AE=Adverse Event CRF=Case Report Form    -   DCF=Data Clarification Form FDA=Food and Drug Administration    -   HIPAA=Health Insurance Portability and Accountability Act of        1996    -   IRB/IEC=Institutional Review Board/Independent Ethics Committee    -   ITT=Intent-to-Treat PP=Per Protocol    -   SAE=Serious Adverse Event    -   UADE=Unanticipated Adverse Device Effect

Example 7: Results and Summary of Phase I Clinical Study Introduction

For this phase-I research study 14 genes within the Geneticure bloodpressure (BP) panel were assessed as they relate to time to BP controland absolute BP values in 99 patients with hypertension. The studydesign utilized a post-hoc patient chart review carried out by twoclinical sites through the direction of RCRI (a third-party clinicalresearch firm) exploring genes important in drug metabolism, renal Na⁺handling, vascular function, and cardiac output (all of which can resultdifferences in BP and response to BP therapy). Although the primary aimwas BP control in response to therapy relative to genetic data, the timeon average, it takes patients to achieve BP control withoutconsideration of genetic information was also determined.

In summary, the study demonstrated that the genes in the Geneticurepanel were predictive of time to BP control in patients withhypertension. In addition, there was an effect of several of the genesbeing predictive of BP taken within the clinic at the time of theresearch study. In addition, mechanistic data was gathered for the genesthat encode the alpha subunit of the epithelial Na⁺ channel (SCNN1A, rs#2228576) and found that SCNN1A was predictive of urinary Na⁺concentration and mean arterial BP.

Methods:

The BP history for patients was collected and the current BP levels weremeasured in patients with controlled hypertension. DNA was collectedusing a buccal swab and analyzed the genes within the Geneticure panel.The study sought to determine if patients with “functional” genotypes ofproteins important in certain drug classes responded better if they weretaking the drug that would inhibit the activity of that protein. As anexample, the beta-1 adrenergic receptor (ADRB1) is important in heartrate control and patients who are on a beta-blocker can demonstrate adrop in BP because of inhibition of this protein. Therefore, one wouldhypothesize that if a patient with a functional protein of the ADRB1 isput on a beta-blocker early, they will demonstrate better BP control(because of a greater drop in heart rate and, therefore BP). This wasassessed according to 14 genes and 3 major classes of BP drugs(diuretic, vasodilator, beta-blocker) and one drug metabolizing enzyme(CYP2D6).

-   Results: Subject Characteristics-   Demographics (n=99)

Variable mean ± SEM Age (yrs)  58 ± 0.8 Sex (% female) 46 Diabetes (%with) 28 ± 4 Weight (kg)  86 ± 1.4 Height (cm) 169 ± 1  BMI (kg/m²) 29.9± 0.4

-   Results: Blood Pressure Data (n=99)

Variable Mean ± SEM Initial SBP (mmHg) 151 ± 2  Initial DBP (mmHg) 91 ±1 Initial MAP (mmHg) 111 ± 1  Lowest SBP in past two years (mmHg) 115 ±1  Lowest DBP in past two years (mmHg) 72 ± 1 Current SBP (mmHg) 134 ±2  Current DBP (mmHg) 82 ± 1 Current MAP (mmHg) 99 ± 1 Time to BPcontrol (months)  22 ± 10 Clinic Visits in the Past two years for HTN 3.6 ± 0.3

-   Results: Current Blood Pressure Therapy Information-   Drug Class Usage (n=99)

Variable mean ± SEM Number of Classes of Drugs for HTN  1.8 ± 0.08Diuretic (% taking) 42 ± 5 ACE Inhibitor (% taking) 62 ± 5 ARB (%taking) 27 ± 5 B-Blocker (% taking) 33 ± 5 Ca⁺ Channel Blocker (%taking) 16 ± 4These data describe the number of different drug classes that thepatients were taking. In addition, we assessed the percent of subjectswho were on drugs within the vasodilator class (ACE-inhibitor and ARB),the cardiac class (B-blocker Ca⁺ channel blocker), and the renal class(diuretic).Time to Control According to Drug Class (n=99)

Months For Control Clinic Visits/2 Years On the Not on the On the Not onthe Drug Class Drug Class Drug Class Drug Class Drug Class Diuretic 39.5± 20.4 7.9 ± 4.2 4.5 ± 0.6 3.0 ± 0.4* ACE Inhibitor 22.2 ± 11.4 22.5 ±16.4 3.1 ± 0.4 4.5 ± 0.6* Antiotensin 32.8 ± 23.1 17.1 ± 9.1  3.9 ± 0.63.5 ± 0.4  Receptor Blocker B-Blocker 24.5 ± 16.9 21.2 ± 12.0 4.9 ± 0.73.1 ± 0.4* Ca+ Channel 9.9 ± 4.5 25.0 ± 11.7 5.1 ± 0.7 3.3 ± 0.4 Blocker *p < 0.05 compared to those patients who were on the class ofdrugs

These data describe the time it took for BP control according to whichclass of drugs the patient was taking. While there are no significantdifferences in months taken for BP control according to drug class,there was an effect of number of clinic visits (specific tohypertension) within the past 2 years according to drug class. Patientsusing beta-blockade and diuretic therapy to control their BP had fewerclinic visits, when compared to those patients not on these therapies.Patients on an ACE-inhibitor had significantly more clinic visits peryear, when compared to patients not on this therapy.

Blood Pressure Control According to Genotypes (n=86)

1. Genes Important in Renal Na⁺ Handling (and Those that areDifferentially Responsive to Diuretic Therapy).

WNK1 (RS# 1159744) On Target Therapy (Diuretic) No Yes Genotype GGC•containing GG C•containing n 26 24 19 15 Systolic 133.7 ± 3.2  133.5 ±2.3  137.8 ± 4.1  132.1 ± 5.2  Blood Pressure (mmHg) Diastolic 79.7 ±2.3 84.6 ± 2.0 88.7 ± 2.7* 79.5 ± 3.7 Blood Pressure Mean 97.7 ± 2.4100.9 ± 1.81 105.1 ± 2.7*  97.1 ± 3.6 Arterial Blood Pressure (mm Hg)Months to  3.6 ± 1.4  4.8 ± 2.6 8.2 ± 5.6 16.5 ± 6.2 BP Control *P <0.05 compared to same genotype not on target therapy.

SLC12A3 (RS# 1529927) On Target Therapy (Diuretic) No Yes Genotype GGC•containing GG C•containing n 45 6 33 2 Systolic Blood 134.2 ± 2.31136.8 ± 5.9 136.4 ± 3.4 128.0 ± 8   Pressure (mmHg) Diastolic Blood 81.8± 1.7  86.5 ± 2.3  85.9 ± 2.5 75.0 ± 5.0 Pressure (mm Hg) Mean Arterial99.3 ± 1.7 103.3 ± 3.1 102.7 ± 2.4 92.3 ± 6.0 Blood Pressure (mm Hg)Months to BP  2.5 ± 0.7 17.7 ± 7   10.5 ± 3.9 42 Control

WNK1 (RS# 2107614) On Target Therapy No Yes Genotype GG C•containing GC•containing n 9 41 7 27 Systolic Blood 130.3 ± 5.6  134.4 ± 2.1  147.7± 7.9 132.1 ± 3.3 Pressure (mmHg) Diastolic Diastolic Blood 80.4 ± 4.782.4 ± 1.6 84.6 ± 4   84.7 ± 2.8 Pressure (mm Hg) Mean Arterial 97.1 ±4.6 99.7 ± 1.6  106 ± 4.4 100.5 ± 2.6 Blood Pressure (mm Hg) Months toBP 10.5 ± 7.2  2.6 ± 0.9  10.5 ± 9.5  13.1 ± 4.8 Control

Alpha Adducin (RS# 4961) On Target Therapy No Yes Genotype GGT•containing GG T•containing n 40 11 24 11 Systolic Blood 134.2 ± 2.3135.0 ± 5.3  135.3 ± 3.9 137.4 ± 5.9 Pressure (mmHg) Diastolic Blood 83.1 ± 1.8 79.7 ± 3.3  85.8 ± 2.9  84.2 ± 4.3 Pressure (mm Hg) MeanArterial 100.2 ± 1.8 98.2 ± 3.6 102.3 ± 2.7 101.9 ± 4.5 Blood Pressure(mm Hg) Months to BP  4.4 ± 1.6  3.6 ± 15.1  10.5 ± 5.2  15.1 ± 6.7Control

Of the four genes explored in the clinical study RS #1159744 (the genethat encodes cytoplasmic serine-threonine kinase that is expressed inthe kidney, WNK-1) was most predictive of response to therapy. Patientswith the C genotype of WNK-1 had the best response to therapydemonstrating 8 mmHg lower DBP, when compared to patients with thisgenotype who were not on diuretic therapy. Subjects who were homozygousfor G for this gene actually had a lower blood pressure if they were noton a diuretic, indicating that they may be benefiting from alternatetherapy. Although just a trend (due to small sample size of the minorallele) the C polymorphism of SLC12A3 also may be predictive of responseto diuretic therapy with patients demonstrating an 11 mmHg drop in DBPwith therapy, compared to the G polymorphism which demonstrated a smallincrease in DBP with therapy.

In addition to this clinical BP data 24-hour urinary and resting BP datawere gathered according to genetic variation of the alpha sub-unit ofthe epithelial Na⁺ channel (SCNN1A, RS #2228576). It was found thatsubjects homozygous for the T variant of SCNN1A demonstrated more Na⁺excretion from the kidneys and they also demonstrated lower meanarterial blood pressure, when compared to genotype groups containing theA variant (See FIGS. 8 and 9).

2. Genes Important in Cardiac Function (and Those that May RespondDifferentially to Beta-Blocker Therapy).

The beta-1 adrenergic receptor (ADRB1) is important in controlling heartrate and cardiac contractility.

Beta-1 Adrenergic Receptor 49 (RS# 1801252) On Target Therapy No Yes(Beta-Blocker) Thr•con- Thr•con- Genotype Ser/Ser taining Ser/Sertaining n 1 55 2 28 Systolic Blood 137 136.4 ± 2.3 136 ± 6  132.4 ± 3.2 Pressure (mmHg) Diastolic Diastolic Blood 86  85.5 ± 1.7 75 ± 3 80.3 ±2.1* Pressure (mm Hg) Mean Arterial 103 102.4 ± 1.7 95 ± 0 97.7 ± 2.1 Blood Pressure (mm Hg) Months to BP N/A  7.0 ± 2.4 N/A 9.4 ± 3.8 Control*P < 0.05 compared to same genotype not on target therapy.

Beta-1 Adrenergic Receptor 389 (RS# 1801253) On Target Therapy (Beta-Blocker) No Yes Genotype GG C•containing GG C•containing n 56 0 30 0Systolic 136.4 ± 2.3 132.6 ± 3.0  Blood Pressure (mmHg) DiastolicDiastolic  85.5 ± 1.7 80.0 ± 2.0* Blood Pressure (mm Hg) Mean 102.4 ±1.7 97.5 ± 1.98 Arterial Blood Pressure (mm Hg) Months to  7.0 ± 2.3 8.8± 3.5 BP Control *P < 0.05 compared to same genotype not on targettherapy.

These data indicate a differential BP response to beta-blocker therapyaccording to genetic variation at position 49 of the ADRB1.Specifically, the inventors found that subjects with the Ser genotype atposition 49 of ADRB1 benefit from beta-blocker therapy with an averagedrop in DBP of 11 mmHg, compared with a drop of 5 mmHg with Thr at thisposition. Therefore, although patients with the Thr polymorphism alsodemonstrated a drop in BP with beta-blocker therapy, the effect was mostpronounced in patients with the Ser polymorphism.

The beta-2 adrenergic receptor (ADRB2) is important in cardiaccontractility, which controls stroke volume, and can influence BPthrough differences in cardiac output.

Beta-2 Adrenergic Receptor 16 (RS# 1042713) On Target Therapy No Yes(B-Blocker) Gly-con- Gly-con- Genotype Arg/Arg taining Arg/Arg taining n5 51 3 27 Systolic Blood 143.6 ± 5.9  135.7 ± 2.4 129 ± 5  133.3 ± 3   Pressure (mmHg) Diastolic Blood 84.0 ± 7.4  85.6 ± 1.8 80.3 ± 6.6 79.9 ±2.1* Pressure (mmHg) Mean Arterial 104.0 ± 6.82 102.3 ± 1.8 96.6 ± 5.997.6 ± 2.1  Blood Pressure (mmHg) Months to BP  7 ± 5    7 ± 2.6  4.3 ±3.8 9.9 ± 4.3 Control *P < 0.05 compared to same genotype not on targettherapy

Beta-2 Adrenergic Receptor 27 (RS# 1042714) On Target Therapy No Yes(B-Blocker) Glu-con- Glu-con- Genotype Gln/Gln taining Gln/Gln taining n6 48 4 24 Systolic Blood 142.0 ± 6.9 134.4 ± 2.2 125.5 ± 5.9 132.7 ±3.6  Pressure (mmHg) Diastolic Blood  82.5 ± 2.7  85.1 ± 1.8  82.5 ± 2.779.5 ± 2.4 Pressure (mmHg) Mean Arterial 102.3 ± 4.1 101.5 ± 1.8 102.3 ±4.1 97.2 ± 2.3 Blood Pressure (mmHg) Months to  8.5 ± 7.8    7 ± 2.8 5.3 ± 2.3  9.6 ± 4.4 BP Control

These data demonstrate a generally favorable response to beta-blockertherapy with both genotype groups. However, the Gly16 genotypedemonstrated a statistically significant difference in BP control if thepatients were on a beta-blocker (drop in DBP of 5 mmHg), when comparedto patients with the Arg16 genotype. Generally, there is a similarpattern for a greater drop in BP with subjects who have the mostfunctional gene that encodes the ADRB2 (Gly at position 16 and Glu atposition 27). There is strong linkage disequilibrium between these twosites (amino acids 16 and 27), so the similar response between the sitesis expected.

Observations on Genetic Variation of Cytochrome P450 2D6 (CYP2D6), whichis Important in Drug Metabolism, Especially of Particular Beta-Blockers.

CYP 2D6 (RS#) On Target Therapy (B-Blocker) No Yes Genotype CCT-Containing CC T-Containing n 35 22 23 7 Systolic 140.6 ± 2.7 128.5 ±3.1  133.4 ± 3.6  130.0 ± 5.7  Blood Pressure (mmHg) Diastolic  86.0 ±2.4 83.2 ± 2.4 79.2 ± 2.1* 82.6 ± 5.3 Blood Pressure (mmHg) Mean 104.2 ±2.3 98.3 ± 2.4 97.3 ± 2.2* 98.4 ± 4.8 Arterial Blood Pressure (mmHg)Months to  7.8 ± 2.9 5.08 ± 3.7 6.25 ± 3.2   16.5 ± 10.8 BP Control *P <0.05 compared to same genotype not on target therapy

These data demonstrate that the CC homozygous group of CYP2D6demonstrates the greatest response to beta-blocker therapy, whencompared to the CT and TT groups. Patients with the CC polymorphism haddemonstrated a 6 mmHg lower DBP and a 7 mmHd lower MAP when onbeta-blocker therapy, compared to this genotype not on beta-blockertherapy. In contrast, patients in the T-containing group (those with theCT and TT genotypes) did not respond to beta-blocker therapy.

3. Genes Important in Vascular Function (and Those that May RespondDifferentially to Vasodilator Therapy).

The following are observations on the genetic variation of theangiotensin gene (encoding a precursor to angiotensin-II, a potentvasoconstrictor, which is converted via angiotensin converting enzyme,ACE) and the responses to various therapies.

Angiotensin (RS# 699) On Target Therapy (Angiotensin Receptor Blocker)No Yes Genotype CC T-containing CC T-containing n 15 50  4 17 SystolicBlood 135.3 ± 2.6 135.1+2.6 135.0 ± 7.4  135.1 ± 4.5 Pressure (mmHg)Diastolic Blood  89.5 ± 2.9  82.6 ± 1.7 77.5 ± 7.8  82.7 ± 3.4 Pressure(mmHg) Mean Arterial 104.7 ± 2.6 100.1 ± 1.7 96.6 ± 7.5 100.2 ± 3.3Blood Pressure (mmHg) Months to  2.7 ± 1.3  6.7 ± 2.7 12  12.4 ± 4.8 BPControl

Angiotensin (RS# 699) On Target Therapy (ACE- Inhibitor) No Yes GenotypeCC T-containing CC T-containing n 6 24 13 43 Systolic 131.7 ± 5.3  133.7± 3.5  136.8 ± 2.7  135.8 ± 2.9 Blood Pressure (mmHg) Diastolic 78.5 ±5.0 80.4 ± 2.1 90.8 ± 3.2*  83.9 ± 2.0 Blood Pressure (mmHg) Mean 96.2 ±4.7   98.2± 106.2 ± 2.8*  101.1 ± 2.0 Arterial Blood Pressure (mmHg)Months to  6.7 ± 3.1  7.7 ± 3.2 2.3 ± 1.7  9.9 ± 3.8 BP Control *P <0.05 compared to same genotype not on target therapy

All Receptor Type-1 (RS# 5186) On Target Therapy (Angiotensin ReceptorBlocker) No Yes Genotype AA C-containing AA C-containing n 39 28 9 11Systolic Blood 137.3 ± 2.6 131.0 ± 3.1  136.3 ± 4.6 134.1 ± 6.5 Pressure (mmHg) Diastolic Blood  85.4 ± 2.1 80.9 ± 2.0  87.6 ± 3.9 77.9± 4.5 Pressure (mmHg) Mean Arterial 102.7 ± 2.0 97.6 ± 2.1 103.8 ± 2.996.6 ± 4.9 Blood Pressure (mmHg) Months to  7.3 ± 2.9  3.8 ± 2.5  13.4 ±7.8 11.9 ± 5.7 BP Control

These data indicate that patients homozygous for the C genotype ofangiotensin may benefit from an angiotensin receptor blocker (ARB). Whenon an ARB, patients with the CC genotype demonstrated a 12 mmHg lowerDBP when compared to patients with this genotype who were not on thistherapy. In contrast, patients in the T-containing group (those with theCT or TT genotypes) did not show a response to ARB therapy. Furthermore,inhibition of ACE (which converts angiotensin-I to angiotensin-II)results in higher BP levels, possibly due to a “build-up” ofangiotensin. Therefore, these data indicate that patients homozygous forC should be given an angiotensin receptor blocker with an ACE inhibitor.

Angiotensin Converting Enzyme (ACE) Genotype and ACE-Inhibition

ACE (RS# 1799752) On Target Therapy (ACE- No Yes Inhibition) Del• Del•Genotype Ins/Ins Containing Ins/Ins Containing n 3 27 12 47 Systolic142.7 ± 11.1 132.3 ± 3.1  129.5 ± 3.5  137.1 ± 2.6 Blood Pressure (mmHg)Diastolic 79.6 ± 6.1 80.0 ± 2.1 80.4 ± 4.4  85.7 ± 1.9* Blood Pressure(mm Hg) Mean 100.7 ± 7.7  97.4 ± 2.1 96.8 ± 3.8 102.8 ± 1.8 ArterialBlood Pressure (mm Hg) Months to  4.7 ± 3.7 8.0 ± 3   7.4 ± 5.8  7.3 ±3.0 BP Control *P < 0.05 compared to same genotype not on targettherapy.

These data indicate that those with the Insertion polymorphism (Ins) ofthe ACE gene will respond best to ACE-inhibition. Patients with the Delpolymorphism actually demonstrated higher DBP with ACE-inhibition, whencompared to this patient group not on ACE-inhibitors.

Observations on Renin Genotype and Angiotensin Receptor Blocker

Renin is a precursor to angiotensin and, therefore, patients with afunctional genotype of renin may benefit from Angiotensin ReceptorBlocker (ARB) therapy because a more functional genotype can lead togreater angiotensin levels which can result in high BP.

Renin (RS# 12750834) On Target Therapy (Angiotensin Receptor Blocker) NoYes Genotype G/G A•containing G/G A•containing n 48 17 14 7 Systolic134.8 ± 2.3 135.9 ± 4.4 136.4 ± 4.6 132.2 ± 7.3  Blood Pressure (mmHg)Diastolic  83.8 ± 1.7  85.1 ± 2.9  82.0 ± 2.9 81.0 ± 7.4 Blood Pressure(mm Hg) Mean 100.8 ± 1.7 102.1 ± 2.9 100.2 ± 3.2 98.1 ± 6.6 ArterialBlood Pressure (mm Hg) Months to  4.75 ± 1.9  8.0 ± 5.8  15.5 ± 5.3* 1.3 ± 0.7 BP Control *P < 0.05 compared to same genotype not on targettherapy.

These data indicate that the functional genotype of renin (A) maybenefit from ARB therapy. Specifically, the BP response to therapy wasnot significant, however, the response to therapy time was pronounced.Patients who have the functional genotype of renin (the AG and AAgenotype groups) demonstrate a much shorter time to BP control, whencompared to patients within this group who do not take this therapy. Incontrast, patients in the GG group demonstrate a longer time to controlif they take this therapy, possibly due to greater response to anotherclass of drugs.

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All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods, devices, and kits described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and the methods and processes are not necessarilyrestricted to the orders of steps indicated herein or in the claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a nucleic acid” or “apolypeptide” includes a plurality of such nucleic acids or polypeptides(for example, a solution of nucleic acids or polypeptides or a series ofnucleic acid or polypeptide preparations), and so forth. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention.

What is claimed:
 1. A method for treating an individual patient havinghypertension, the method comprising administering to the individualpatient a first line therapy antihypertensive drug selected from thegroup consisting of a diuretic drug, a vasodilator drug, and a betablocker drug, wherein the vasodilator drug is an ACE inhibitor drug oran angiotensin II receptor blocker drug, and the diuretic drug is athiazide diuretic drug, a loop diuretic drug, or an amiloride potassiumsparing drug; the method of administering comprising the steps of:determining the individual patient's genotype and corresponding peptidesequences for each of the following polymorphic nucleotide andpolypeptide sequences: rs1159744; rs2107614; rs4961; rs1529927;rs3892097; rs1801253; rs1801252; rs12750834; rs5186; rs1799752; rs699;rs2228576; rs1042714; rs1042713; ADRB1 (β₁AR) polypeptide with serine atposition 49; ADRB1 (β₁AR) polypeptide with arginine at position 389;ADRB2 (β₂AR) polypeptide with glycine at position 16; ADRB2 (β₂AR)polypeptide with glutamic acid at position 27; angiotensin polypeptidewith threonine at position 268; Na⁺ channel (SCNN1A) polypeptide withthreonine at position 663; and sodium chloride co-transporter (SLC12A3)polypeptide with alanine at position 264; and administering to theindividual patient a first line therapy antihypertensive drug accordingone of the following procedures a, b, c, d, e, or f: a) administeringthe beta-blocker drug as the first line therapy antihypertensive drugand not administering the diuretic drug or the vasodilator drug, whereinthe individual patient's genotype and peptide sequences have beendetermined to be homozygous for: 1) an ADRB1 (β₁AR) polypeptide withserine at position 49; 2) an ADRB1 nucleic acid with adenine at thevariable position of rs1801252; 3) an ADRB1 (β₁AR) polypeptide witharginine at position 389; 4) an ADRB1 nucleic acid with cytosine atvariable position of rs1801253; 5) an ADRB2 (β₂AR) polypeptide withglycine at position 16; 6) an ADRB2 nucleic acid with guanine at thevariable position of rs1042713; 7) an ADRB2 (β₂AR) polypeptide withglutamic acid at position 27; or 8) an ADRB2 nucleic acid with guanineat the variable position of rs1042714; and not homozygous for: i)adenine at the variable position of rs3892097; ii) thymine at thevariable position of rs4961; iii) thymine at the variable position ofrs1529927; iv) a cytosine at the variable position of rs1159744; and, v)a cytosine at the variable position of rs2107614, b) administeringatenolol or carvedilol as the first line therapy antihypertensive drugwherein the patient's genotype has been determined to be homozygousfor: 1) adenine at the variable position of rs3892097; and nothomozygous for: i) thymine at the variable position of rs4961; ii)thymine at the variable position of rs1529927; iii) a cytosine at thevariable position of rs1159744; and, iv) a cytosine at the variableposition of rs2107614, c) administering an ACE inhibitor drug as thefirst line therapy antihypertensive drug and not administering thediuretic drug or the beta blocker drug, wherein the individual patient'sgenotype and polypeptide sequences have been determined to be homozygousfor: 1) an angiotensin polypeptide with a threonine at position 268; 2)an angiotensin nucleic acid with cytosine at the variable position ofrs699; or 3) a nucleic acid with a deletion in rs1799752; and nothomozygous for: i) thymine at the variable position of rs4961; ii)thymine at the variable position of rs1529927; iii) a cytosine at thevariable position of rs1159744; and, iv) a cytosine at the variableposition of rs2107614, d) administering an angiotensin II receptorinhibitor blocker drug as the first line therapy antihypertensive drugand not administering the diuretic drug, beta blocker drug or ACEinhibitor drug, wherein the individual patient's genotype has beendetermined to be homozygous for: 1) cytosine at the variable position ofrs5186; or 2) cytosine at the variable position of rs12750834; and nothomozygous for: i) thymine at the variable position of rs4961; ii)thymine at the variable position of rs1529927; iii) a cytosine at thevariable position of rs1159744; and iv) a cytosine at the variableposition of rs2107614; e) administering the diuretic drug as the firstline therapy antihypertensive drug and not administering the vasodilatordrug or the beta blocker drug, wherein the individual patient's genotypeand peptide sequences have been determined to be homozygous for: 1) aNa⁺ channel (SCNN1A) polypeptide with threonine at position 663; 2) anadenine at the variable position of rs2228576; 3) an adducin polypeptidewith tryptophan at position 460; 4) a thymine at the variable positionof rs4961; 5) a sodium (Na⁺) chloride (Cl⁻) co-transporter (SLC12A3)polypeptide with an alanine at position 264; 6) a cytosine at thevariable position of rs1529927; 7) a cytosine at the variable nucleotideposition of rs1159744; 8) a cytosine at the variable position ofrs2107614; or 9) any combination thereof; or f) administering a loopdiuretic drug as the first line therapy antihypertensive drug and notadministering the vasodilator drug, or the beta blocker drug, whereinthe individual patient's nucleotide and peptide sequences has beendetermined to not be homozygous for the polymorphic nucleotide andpeptide sequence variants at procedures a) 1-8, b) 1, c) 1-3, and d) 1-2and heterozygous for the polymorphic nucleotide and peptide sequencevariants at procedures e) 1-6.
 2. A method according to claim 1 whereinthe individual patient exhibits an improved blood pressure response tothe first line therapy antihypertensive drug relative to the responsethe individual patient would exhibit as a result of administration of anantihypertensive drug which is different than the drug administeredaccording to claim
 1. 3. A method according to claim 1, furthercomprising obtaining an analysis of said nucleotide polymorphic andpolypeptide sequences in a sample of the patient's fluid or tissue.
 4. Amethod according to claim 3, wherein the nucleotide polymorphic andpolypeptide sequence analyses are conducted sequentially.
 5. A methodaccording to claim 3, wherein the nucleotide polymorphic and polypeptidesequence analyses are conducted simultaneously.
 6. A method according toclaim 1, further comprising administering to the patient a secondantihypertensive drug after at least one month of administration of thefirst line drug, wherein the second drug is selected from the groupconsisting of a diuretic, a beta-blocker, and a vasodilator, and whereinthe second drug is not the same as the first line drug.
 7. A methodaccording to claim 1 wherein the diuretic is furosemide, a thiazide, acarbonic anhydrase inhibitor, an aldosterone antagonist, spironolactone,eplerenone, potassium canreonate, amiloride, triamterene, aldactone,acetazolamide, amiloride, bumetanide, chlorothalidone, chlorothiazide,ethacrynic acid, furosemide, glycerin, hydrochlorothiazide,hydroflumthiazide, indapamide, isosorbide, mannitol, methazolamide,methylchlothiazide, metolazone, dichlorphenamide, spironolactone,torsemide, triamterene, urea, altizide, trichlormethazide,triflumethazide, bemetizide, cyclothiazide, methylchlothiazide,azosemide, butizide, bendroflumethazide, cyclopenthiazide,benzclortriazide, polythiazide, hydroflumethazide, benzthiazide,ethiazide, penflutizide, or any combination thereof.
 8. A methodaccording to claim 1 wherein the ACE inhibitor is enalapril, lisinopril,captopril alacipril, benazapril, cilazapril, delapril, fosinopril,perindopril, quinapril, ramipril, moveltipril, spirapril, ceronapril,imidapril, temocapril, trandolopril, utilbapril, zofenopril, CV5975, EMD56855, libenzapril, zalicipril, HOE065, MDL 27088, AB47, DU 1777, MDL27467A, or Y23785.
 9. A method according to claim 1 wherein theangiotensin II receptor antagonist is selected from losartan, valsartan,candesartan, irbesartan, olmesartan, or any combination thereof.
 10. Amethod according to claim 1 wherein the beta blocker is propranolol,atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartrate.